Chemokine uses; compositions; methods

ABSTRACT

Agonists or antagonists of MIP-3α, and various methods of use in dermatological and related applications are provided. In particular, the method makes use of fact that the MIP-3α chemokine is specifically capable of inducing migration of a skin cell subset.

[0001] The present filing is a conversion to U.S. utility patentapplication from provisional U.S. Ser. No. 60/118,335, filed Feb. 3,1999, which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates generally to methods of using variouschemokine related compositions, more particularly, to methods oftreating skin diseases or conditions associated with misregulation ofthe chemokine MIP-3α, a ligand for the CCR6 chemokine receptor.

BACKGROUND

[0003] The immune system consists of a wide range of distinct celltypes, each with important roles to play. See Paul (ed. 1997)Fundamental Immunology 4th ed., Raven Press, New York. The lymphocytesoccupy central stage because they are the cells that determine thespecificity of immunity, and it is their response that orchestrates theeffector limbs of the immune system. Two broad classes of lymphocytesare recognized: the B lymphocytes, which are precursors of antibodysecreting cells, and the T (thymus-dependent) lymphocytes. T lymphocytesexpress important regulatory functions, such as the ability to help orinhibit the development of specific types of immune response, includingantibody production and increased microbicidal activity of macrophages.Other T lymphocytes are involved in direct effector functions, such asthe lysis of virus infected-cells or certain neoplastic cells.

[0004] The chemokines are a large and diverse superfamily of proteins.The superfamily is subdivided into two classical branches, based uponwhether the first two cysteines in the chemokine motif are adjacent(termed the “C—C” branch), or spaced by an intervening residue(“C—X—C”). A more recently identified branch of chemokines lacks twocysteines in the corresponding motif, and is represented by thechemokines known as lymphotactins. Another recently identified branchhas three intervening residues between the two cysteines, e.g., CX3Cchemokines. See, e.g., Schall and Bacon (1994) Current Opinion inImmunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy &Immunol. 109:97-109.

[0005] Many factors have been identified which influence thedifferentiation process of precursor cells, or regulate the physiologyor migration properties of specific cell types. These observationsindicate that other factors exist whose functions in immune functionwere heretofore unrecognized. These factors provide for biologicalactivities whose spectra of effects may be distinct from knowndifferentiation or activation factors. The absence of knowledge aboutthe structural, biological, and physiological properties of theregulatory factors which regulate cell physiology in vivo prevents themodulation of the effects of such factors. Thus, medical conditionswhere regulation of the development or physiology of relevant cells isrequired remain unmanageable.

SUMMARY OF THE INVENTION

[0006] The present invention is based, in part, upon the surprisingdiscovery that the MIP-3( chemokine is expressed in inflamed skin cells.The chemokine is the ligand for the CCR6 receptor. See Greaves, et al.(1997) J. Expt'l Med. 186:837-844. Both the ligand and receptor areexpressed at essentially undetectable levels in normal skin, while bothare highly upregulated in inflamed skin.

[0007] The present invention provides methods of modulating migration ofa cell within or to the skin of a mammal comprising administering to themammal an effective amount of: an antagonist of MIP-3α; an agonist ofMIP-3α, and antagonist of CCR6; or an agonist of CCR6. Typically, themigration is within the skin; or may be chemotactic or chemokinetic. Inpreferred embodiments, the administering is systemic, local, topical,subcutaneous, intracutaneous, or transdermal. Often, the cell is a Tcell, B cell, dendritic cell, or dendritic cell precursor. In otherembodiments, the cell is a T cell, or moves into the dermal and/orepidermal layers of the skin.

[0008] In other embodiments, the administering is of an antagonist ofMIP-3α. Generally, the antagonist is selected from: a mutein of naturalMIP-3α; an antibody which neutralizes MIP-3α; or an antibody which bindsto CCR6. In various embodiments, the mammal is subject to a skin diseaseor condition, including one selected from cancer, cancer metastasis,skin transplant, or skin graft. Often, the antagonist is administered incombination with an antibiotic, antifungal, antiviral, or analgesic; ormay be with an immune suppressive therapeutic, anti-inflammatory drug,growth factor, or immune adjuvant.

[0009] In another embodiment, the administering is with a primateMIP-3α. Often, the modulating is attracting the cell, e.g., to a site ofcutaneous lesion. The primate MIP-3α may be administered in combinationwith an antibiotic, antifungal, antiviral, or analgesic; or with avasodilator, growth factor, cytokine, anti-inflammatory drug, or immuneadjuvant.

[0010] Alternatively, the invention provides a method of purifying apopulation of cells, the method comprising contacting the cells withMIP-3α, thereby resulting in the identification of cells expressing areceptor for MIP-3α. In certain embodiments, the receptor is CCR6, orthe contacting results in specific migration of the cells to a site forpurification, e.g., through pores of a membrane.

DETAILED DESCRIPTION OF THE INVENTION

[0011] Outline

[0012] I. General

[0013] II. Chemokine Agonists and Antagonists

[0014] A. MIP-3α and Variants

[0015] B. Antibodies

[0016] C. Other Molecules

[0017] III. Immunoassays

[0018] IV. Uses

[0019] I. General

[0020] The invention is based, in part, on the surprising discovery thatthe chemokine MIP-3α has been implicated in roles in skin-immunity. Inparticular, MIP-3α has been identified as a ligand for the chemokinereceptor designated CCR6. Both MIP-3α and CCR6 expression areundetectable in normal skin, while both are highly upregulated ininflamed skin samples.

[0021] The skin consists of a surface layer of epithelium called theepidermis and an underlying layer of connective tissue called thedermis. Under the dermis is a layer which contains large amounts ofadipose tissue, the hypodermis. The skin serves a variety of functions,and variations in the character of the dermis and epidermis occuraccording to functional demands. The appendages of the skin, hair,nails, and sweat and sebaceous glands, are such local specializations ofthe epidermis. Together, the skin and its appendages form theintegument. See, e.g., Fitzpatrick, et al. (eds. 1993) Dermatology inGeneral Medicine 4th ed., McGraw-Hill, NY; Bos (ed. 1989) Skin ImmuneSystem CRC Press, Boca Raton, Fla.; Callen (1996) General PracticeDermatology Appleton and Lange; Rook, et al. (eds. 1998) Textbook ofDermatology Blackwell; Habifor and Habie (1995) Clinical Dermatology: AColor Guide to Diagnosis and Therapy Mosby; and Grob (ed. 1997)Epidemiology, Causes and Prevention of Skin Diseases Blackwell.

[0022] The epidermis consists of many different cell types in variousproportions. The most prevalent cell type is keratinocytes, which makeup some 95% of the cells. Cells in the 1-2% range include melanocytesand Langerhans cells. The Langerhans cells are particularly importantbecause they trap antigens that have penetrated the skin, and transportantigens to regional lymph nodes. A small population of γδ T cells canalso reside in the epidermis.

[0023] The dermis varies in thickness in different regions of the body.It is tough, flexible, and highly elastic, and consists of a feltwork ofcollagen fibers with abundant elastic fibers. The connective tissue isarranged into deep reticular and superficial papillary layers.

[0024] The chemokines are a sub-family of chemoattractant cytokines thatwere classically characterized by their ability to mediate leukocytetrafficking or migration by binding to specific G-protein-linked seventransmembrane spanning receptors, or GPCRs. Chemokines are divided intofour groups based on the primary sequence of the first two cysteines:the CXC, CC, C, and CX3C families. The CXC and C families are effectivepredominantly on neutrophils and lymphocytes, respectively. The CCchemokines are preferentially effective on macrophages, lymphocytes, andeosinophils.

[0025] The chemokine MIP-3α, from human, mouse, and rat, has beendescribed earlier. See, e.g., human, GenBank HSU77035; mouse, GenBankAF099052; rat, GenBank U90447; Li and Adams, WO 94-US9484; and Wilde, etal. WO 9616979; each of which is incorporated herein by reference forall purposes. The sequences are provided in Table 1. TABLE 1 A primate,human, MIP-3α, nucleic acid sequence (SEQ ID NO: 1) and amino acidsequence (SEQ ID NO: 2). Coding sequence begins at about nucleotide 1and ends at about 288; CC motif at amino acid residues 6-7. A signalsequence is indicated, but based upon related genes; slightly differentprocessing may occur in different cell types. atg tgc tgt acc aag agtttg ctc ctg gct gct ttg atg tca gtg ctg 48 Met Cys Cys Thr Lys Ser LeuLeu Leu Ala Ala Leu Met Ser Val Leu    −25                 −20                 −15 cta ctc cac ctc tgc ggcgaa tca gaa gca gca agc aac ttt gac tgc 96 Leu Leu His Leu Cys Gly GluSer Glu Ala Ala Ser Asn Phe Asp Cys−10                  −5              −1   1               5 tgt ctt ggatac aca gac cgt att ctt cat cct aaa ttt att gtg ggc 144 Cys Leu Gly TyrThr Asp Arg Ile Leu His Pro Lys Phe Ile Val Gly             10                  15                  20 ttc aca cgg cagctg gcc aat gaa ggc tgt gac atc aat gct atc atc 192 Phe Thr Arg Gln LeuAla Asn Glu Gly Cys Asp Ile Asn Ala Ile Ile         25                  30                  35 ttt cac aca aag aaaaag ttg tct gtg tgc gca aat cca aaa cag act 240 Phe His Thr Lys Lys LysLeu Ser Val Cys Ala Asn Pro Lys Gln Thr     40                  45                  50 tgg gtg aaa tat att gtgcgt ctc ctc agt aaa aaa gtc aag aac atg 288 Trp Val Lys Tyr Ile Val ArgLeu Leu Ser Lys Lys Val Lys Asn Met 55                  60                  65                  70taa                                                             291Table 1 (continued): A murine, mouse, MIP-3α chemokine nucleic acidsequence (SEQ ID NO: 3) and corresponding amino acid sequence (SEQ IDNO: 4). SignalP software predicts a cleavage between A1a(−1) and Ser1;but the actual cleavage may be on either side by a residue or so. atggcc tgc ggt ggc aag cgt ctg ctc ttc ctt gct ttg gca tgg gta 48 Met AlaCys Gly Gly Lys Arg Leu Leu Phe Leu Ala Leu Ala Trp Val        25                 −20                 −15 ctg ctg gct cac ctctgc agc cag gca gaa gca agc aac tac gac tgt 96 Leu Leu Ala His Leu CysSer Gln Ala Glu Ala Ser Asn Tyr Asp Cys    −10                  −5              −1   1               5 tgc ctctcg tac ata cag acg cca ctt cct tcc aga gct att gtg ggt 144 Cys Leu SerTyr Ile Gln Thr Pro Leu Pro Ser Arg Ala Ile Val Gly                 10                  15                  20 ttc aca agacag atg gcc gat gaa gct tgt gac att aat gct atc atc 192 Phe Thr Arg GlnMet Ala Asp Glu Ala Cys Asp Ile Asn Ala Ile Ile             25                  30                  35 ttt cac acg aagaaa aga aaa tct gtg tgc gct gat cca aag cag aac 240 Phe His Thr Lys LysArg Lys Ser Val Cys Ala Asp Pro Lys Gln Asn         40                  45                  50 tgg gtg aaa agg gctgtg aac ctc ctc agc cta aga gtc aag aag atg 288 Trp Val Lys Arg Ala ValAsn Leu Leu Ser Leu Arg Val Lys Lys Met     55                  60                  65taa                                                             291Table 1 (continued): A murine, rat, MIP-3α chemokine nucleic acidsequence (SEQ ID NO: 5) and corresponding amino acid sequence (SEQ IDNO: 6). SignalP software predicts a cleavage between Ala(−1) and Ala1;but the actual cleavage may be on either side by a residue or so. atggcc tgc aag cat ctg ccc ttc ctg gct ttg gcg ggg gta ctg ctg 48 Met AlaCys Lys His Leu Pro Phe Leu Ala Leu Ala Gly Val Leu Leu−25                 20                 −15                 −10 gct tacctc tgc agc cag tca gaa gca gca agc aac ttt gac tgc tgc 96 Ala Tyr LeuCys Ser Gln Ser Glu Ala Ala Ser Asn Phe Asp Cys Cys                 −5              −1   1               5 ctc acg tac acaaag aac gtg tat cat cat gcg aga aat ttt gtg ggt 144 Leu Thr Tyr Thr LysAsn Val Tyr His His Ala Arg Asn Phe Val Gly         10                  15                  20 ttc aca aca cag atggcc gac gaa gct tgt gac att aat gct atc atc 192 Phe Thr Thr Gln Met AlaAsp Glu Ala Cys Asp Ile Asn Ala Ile Ile     25                  30                  35 ttt cac ctg aag tcg aaaaga tcc gtg tgc gct gac cca aag cag atc 240 Phe His Leu Lys Ser Lys ArgSer Val Cys Ala Asp Pro Lys Gln Ile 40                  45                  50                  55 tgg gtgaaa agg att ttg cac ctc ctc agc cta aga acc aag aag atg 288 Trp Val LysArg Ile Leu His Leu Leu Ser Leu Arg Thr Lys Lys Met                 60                  65                  70taa                                                             291

[0026] TABLE 2 Nucleotide sequence (5′ to 3′) of primate, human,chemokine receptor, CCR6, and the corresponding amino acid sequence(amino to carboxy), see SEQ ID NO: 7 and 8. Nucleotide 579 may be A, C,G, or T, and the codon may code for His or Gln. atg ttt tcg act cca gtgaag att att ttg tgt cag tca ata ctt cat 48 Met Phe Ser Thr Pro Val LysIle Ile Leu Cys Gln Ser Ile Leu His  1               5                  10                  15 att act cagttg att ctg aga tgt tac tgt gct cct tgc agg agg tca 96 Ile Thr Gln LeuIle Leu Arg Cys Tyr Cys Ala Pro Cys Arg Arg Ser             20                  25                  30 ggc agt tct ccaggc tat ttg tac cga att gcc tac tcc ttg atc tgt 144 Gly Ser Ser Pro GlyTyr Leu Tyr Arg Ile Ala Tyr Ser Leu Ile Cys         35                  40                  45 gtt ctt ggc ctc ctgggg aat att ctg gtg gtg atc acc ttt gct ttt 192 Val Leu Gly Leu Leu GlyAsn Ile Leu Val Val Ile Thr Phe Ala Phe     50                  55                  60 tat aag aag gcc agg tctatg aca gac gtc tat ctc ttg aac atg gcc 240 Tyr Lys Lys Ala Arg Ser MetThr Asp Val Tyr Leu Leu Asn Met Ala 65                  70                  75                  80 att gcagac atc ctc ttt gtt ctt act ctc cca ttc tgg gca gtg agt 288 Ile Ala AspIle Leu Phe Val Leu Thr Leu Pro Phe Trp Ala Val Ser                 85                  90                  95 cat gcc actggt gcg tgg gtt ttc agc aat gcc acg tgc aag ttg cta 336 His Ala Thr GlyAla Trp Val Phe Ser Asn Ala Thr Cys Lys Leu Leu            100                 105                 110 aaa ggc atc tatgcc atc aac ttt aac tgc ggg atg ctg ctc ctg act 384 Lys Gly Ile Tyr AlaIle Asn Phe Asn Cys Gly Met Leu Leu Leu Thr        115                 120                 125 tgc att agc atg gaccgg tac atc gcc att gta cag gcg act aag tca 432 Cys Ile Ser Met Asp ArgTyr Ile Ala Ile Val Gln Ala Thr Lys Ser    130                 135                 140 ttc cgg ctc cga tcc agaaca cta ccg cgc agc aaa atc atc tgc ctt 480 Phe Arg Leu Arg Ser Arg ThrLeu Pro Arg Ser Lys Ile Ile Cys Leu145                 150                 155                 160 gtt gtgtgg ggg ctg tca gtc atc atc tcc agc tca act ttt gtc ttc 528 Val Val TrpGly Leu Ser Val Ile Ile Ser Ser Ser Thr Phe Val Phe                165                 170                 175 aac caa aaatac aac acc caa ggc agc gat gtc tgt gaa ccc aag tac 576 Asn Gln Lys TyrAsn Thr Gln Gly Ser Asp Val Cys Glu Pro Lys Tyr            180                 185                 190 can act gtc tcggag ccc atc agg tgg aag ctg ctg atg ttg ggg ctt 624 Xaa Thr Val Ser GluPro Ile Arg Trp Lys Leu Leu Met Leu Gly Leu        195                 200                 205 gag cta ctc ttt ggtttc ttt atc cct ttg atg ttc atg ata ttt tgt 672 Glu Leu Leu Phe Gly PhePhe Ile Pro Leu Met Phe Met Ile Phe Cys    210                 215                 220 tac acg ttc att gtc aaaacc ttg gtg caa gct cag aat tct aaa agg 720 Tyr Thr Phe Ile Val Lys ThrLeu Val Gln Ala Gln Asn Ser Lys Arg225                 230                 235                 240 cac aaagcc atc cgt gta atc ata gct gtg gtg ctt gtg ttt ctg gct 768 His Lys AlaIle Arg Val Ile Ile Ala Val Val Leu Val Phe Leu Ala                245                 250                 255 tgt cag attcct cat aac atg gtc ctg ctt gtg acg gct gct aat ttg 816 Cys Gln Ile ProHis Asn Met Val Leu Leu Val Thr Ala Ala Asn Leu            260                 265                 270 ggt aaa atg aaccga tcc tgc cag agc gaa aag cta att ggc tat acg 864 Gly Lys Met Asn ArgSer Cys Gln Ser Glu Lys Leu Ile Gly Tyr Thr        275                 280                 285 aaa act gtc aca gaagtc ctg gct ttc ctg cac tgc tgc ctg aac cct 912 Lys Thr Val Thr Glu ValLeu Ala Phe Leu His Cys Cys Leu Asn Pro    290                 295                 300 gtg ctc tac gct ttt attggg cag aag ttc aga aac tac ttt ctg aag 960 Val Leu Tyr Ala Phe Ile GlyGln Lys Phe Arg Asn Tyr Phe Leu Lys305                 310                 315                 320 atc ttgaag gac ctg tgg tgt gtg aga agg aag tac aag tcc tca ggc 1008 Ile Leu LysAsp Leu Trp Cys Val Arg Arg Lys Tyr Lys Ser Ser Gly                325                 330                 335 ttc tcc tgtgcc ggg agg tac tca gaa aac att tct cgg cag acc agt 1056 Phe Ser Cys AlaGly Arg Tyr Ser Glu Asn Ile Ser Arg Gln Thr Ser            340                 345                 350 gag acc gca gataac gac aat gcg tcg tcc ttc act atg tga 1098 Glu Thr Ala Asp Asn Asp AsnAla Ser Ser Phe Thr Met        355                 360                 365

[0027] In contrast to naive lymphocytes, memory/effector lymphocytes canaccess non-lymphoid effector sites and display restricted, oftentissue-selective, migration behavior. This results in the presence ofsuch lymphocytes in the peripheral tissues, e.g., outside of thelymphatic and blood volume.

[0028] Both human and mouse MIP-3α are detected in lymph nodes,appendix, PBL, fetal liver, fetal lung, and various cell lines. See,e.g., Rossi, et al. (1997) J. Immunol. 158:1033-1036; Hieshima, et al.(1997) J. Biol. Chem. 272:5846-5853; Baba, et al. (1997) J. Biol. Chem.272:14893-14898; and Imai, et al. (1997) J. Biol. Chem.272:1503.6-15042. The expression in the Langerhans islets suggests arole in skin functions. The data is consistent with MIP-3α as a productof activated monocytes, and is preferentially expressed in inflamedtissue. This distribution would suggest that MIP-3α may have a role inattracting memory T cells, and skin dendritic cells (Langerhans cells)and their precursors. These results suggest an important role for MIP-3αin recruitment of T cells and dendritic cells to peripheral cutaneoussites.

[0029] Chemokine receptors are members of the G protein coupled receptorfamily. See, e.g., Yoshie, et al. (1997) J. Leukoc. Biol. 62:634-644.CCR6 expression has been reported in Greaves, et al. (1997) J. Expt'lMed. 186:837-844; and Liao, et al. (1999) J. Immunol. 162:186-194.Northern blot data showed expression predominantly in the spleen, withlesser amounts in thymus, testis, small intestine, and peripheral blood.Additional transcripts were detected in spleen. Transcripts were notdetected in the TF-1, Jurkat, MRC5, JY, and U937 cell lines. Messageseems not to be abundantly expressed in the lymphoid lineage,particularly in, e.g., libraries made from cells made from dendriticcell cultures derived from cells selected on the basis of CD1aexpression. Expression is lower in DC generated from monocytes.

[0030] Another study showed CCR6 was expressed on memory T cells,including most α4β7 memory cells and cutaneous lymphocyte-associatedantigen expressing cells, and on B cells. Chemotaxis of T cells toMIP-3α was limited to memory cells. See Liao, et al. (1998) J. Immunol.162:186-194. Antiserum detected CCR6 on CD34+ bone marrow deriveddendritic cells.

[0031] Having identified the MIP-3α as a skin related chemokine, it willfind use in affecting medical abnormalities of the skin. Common skindisorders involving the immune system include psoriasis, skin cancers,carcinomas, inflammation, allergies dermatitis, wound healing,infections (both microbial and parasitic), and many others. See, e.g.,The Merck Manual, particularly the chapter on dermatologic disorders.These therapeutics may have useful effects on growth or health ofappendages of the skin, including, e.g., hair, nails, and sweat andsebaceous glands.

[0032] Psoriasis is a chronic inflammatory skin disease that isassociated with hyperplastic epidermal keratinocytes and infiltratingmononuclear cells, including T cells, neutrophils and macrophages.Because of this highly mixed inflammatory picture and the resultingcomplex interrelationships between these different cells, it has beenvery difficult to dissect the mechanisms that underlie the induction andprogression of the disease.

[0033] This view of psoriasis also implies that although dormantautoreactive T cells may pre-exist in susceptible individuals, anenvironmental stimulus is necessary to trigger disease induction. Othersbelieve that the immune system plays only a minor modulatory role in thedisease process and that hyperproliferation of keratinocytes is in factthe initiating event in a genetically susceptible host. Research intothe pathogenesis of psoriasis has long been hindered by the lack ofsuitable animal models.

[0034] There is growing data indicating that T cells and notkeratinocytes are the primary pathogenic component in the disease. Theobservations herein provide evidence to support the concept thatpsoriasis-like conditions can indeed result from unregulated T cellresponses.

[0035] Skin cancers such as basal cell and squamous cell carcinoma areamong the most common malignancies. See, e.g., Miller and Maloney (eds.1997) Cutaneous Oncology: Pathophysiology, Diagnosis, and ManagementBlackwell; Emmett and Orourke (1991) Malignant Skin Tumours ChurchillLivingstone; Friedman (1990) Cancer of the Skin Saunders. Most of thosetumors arise in sun exposed areas of the skin. Immune regulation orclearance of such tumors may depend upon function Qf the skin immunesystem. Cells which effect such may be compromised by localmisregulation or suppression. The MIP-3α or antagonists may break atemporary homeostasis which suppresses normal immune response, therebyleading to activation of proper regulatory and immune pathways.

[0036] Dermatitis is a superficial inflammation of the skin,characterized by vesicles (when acute), redness, edema, oozing,crusting, scaling, and/or itching. See, e.g., Lepoittevin (ed. 1998)Allergic Contact Dermatitis: The Molecular Basis Springer-Verlag;Rietschel and Fowler (eds. 1995) Fisher's Contact Dermatitis Lippincott;and Rycroft, et al. (eds. 1994) Textbook of Contact DermatitisSpringer-Verlag. The term eczematous dermatitis is often used to referto a vesicular dermatitis. Dermatitis may accompany various immunedeficiency conditions or diseases, inborn metabolic disorders, ornutritional deficiency diseases. Certain of the symptoms of suchconditions may be treated using the present invention.

[0037] Pruritus is a sensation that the patient attempts to relieve byscratching. See, e.g., Fleischer and Fleischer (1998) The ClinicalManagement of Itching: Therapeutic Protocols for Pruritus Parthenon.Many parasitic or infectious conditions may result in those symptoms,which conditions may be cleared by proper reactivation or suppression ofimmune functions in the skin. Likewise with various allergic or otherimmune reactions to exposure to various allergic or inflammatoryantigens.

[0038] II. Chemokine Agonists and Antagonists

[0039] Mammalian MIP-3α chemokines were described previously in U.S.Ser. No. 08/887,977, which describes various migratory assays. Variousagonists and antagonists of the natural ligands can be produced. Themigration assays may-take advantage of the movement of cells throughpores in membranes. Chemotaxis may be measured thereby. Alternatively,chemokinetic assays may be developed, which measure the induction ofkinetic movement, not necessarily relative to a gradient, per se.

[0040] A. MIP-3α and Variants

[0041] MIP-3α agonists will exhibit some or all of the signalingfunctions of MIP-3α e.g., binding, inducing a Ca++ flux, andchemoattracting appropriate receptor bearing cells. Various mammalianMIP-3α sequences may be evaluated to determine what residues areconserved across species, suggesting what residues may be changedwithout dramatic effects on biological activity. Alternatively,conservative substitutions are likely to retain biological activity,thus leading to variant forms of the chemokine which will retain agonistactivity. Standard methods for screening mutant or variant MIP-3αpolypeptides will determine what sequences will be useful therapeuticagonists.

[0042] In addition, certain nucleic acid expression methods may beapplied. For example, in skin graft contexts, it may be useful totransfect the grafts with nucleic acids which will be expressed, asappropriate. Various promoters may be operably linked to the gene,thereby allowing for regulated expression. Antisense constructs mayprevent expression of the ligand or receptor.

[0043] Alternatively, antagonist activity may be tested or screened for.Tests for ability to antagonize chemoattractant activity can bedeveloped using assays as described below. Various ligand homologs canbe created which retain receptor binding capacity, but lack signalingcapability, thus serving as competitive binding molecules. Smallmolecules may also be screened for ability to antagonize MIP-3αfunction, e.g., chemoattraction, receptor binding, Ca++ flux, and othereffects mediated by MIP-3α. See generally Gilman, et al. (eds. 1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8thEd., Pergamon Press; Remington's Pharmaceutical Sciences, 17th ed.(1990), Mack Publishing Co., Easton, Pa., each of which is incorporatedherein by reference.

[0044] B. Antibodies

[0045] The present invention provides for the use of an antibody orbinding composition which specifically binds to MIP-3α, preferablymammalian, e.g., primate, human, cat, dog, rat, or mouse, andneutralizes the ability of the chemokine to mediate its signal.Antibodies can be raised to various MIP-3α proteins, includingindividual, polymorphic, allelic, strain, or species variants, andfragments thereof, either in their naturally occurring (full-length)forms or in their recombinant forms. Additionally, antibodies can beraised to MIP-3α or polypeptides in both their native (or active) formsor in their inactive, e.g., denatured, forms, which may neutralizeligand capacity to mediate its signal. Antibodies may block theinteraction of the ligand with its receptor.

[0046] Alternatively, receptor antagonists may be produced by makingantibodies which bind to the receptor and block ligand binding. With theidentification of the CCR6 as a receptor for the cytokine, antibodies tothe receptor may be selected for those which block the binding of, orsignaling induced by, ligand.

[0047] A number of immunogens may be selected to produce antibodiesspecifically reactive, or selective for binding, with MIP-3α or CCR6proteins. Recombinant protein is a preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Naturally occurringprotein, from appropriate sources, e.g., primate, rodent, etc., may alsobe used either in pure or impure form. Synthetic peptides, made usingthe MIP-3α or CCR6 protein sequences described herein, may also be usedas an immunogen for the production of antibodies. Recombinant proteincan be expressed and purified in eukaryotic or prokaryotic cells asdescribed, e.g.,. in Coligan, et al. (eds. 1995 and periodicsupplements) Current Protocols in Protein Science John Wiley & Sons, NewYork, N.Y.; and Ausubel, et al (eds. 1987 and periodic supplements)Current Protocols in Molecular Biologvy, Greene/Wiley, New York, N.Y.Naturally folded or denatured material, perhaps expressed on cellsurfaces, can be used, as appropriate, for producing antibodies. Eithermonoclonal or polyclonal antibodies may be generated, e.g., forsubsequent use in immunoassays to measure the protein, or forimmunopurification methods.

[0048] Methods of producing polyclonal antibodies are well known tothose of skill in the art. Typically, an immunogen, preferably apurified protein, is mixed with an adjuvant and animals are immunizedwith the mixture. The animalls immune response to the immunogenpreparation is monitored by taking test bleeds and determining the titerof reactivity to, e.g., the MIP-3α protein or polypeptide of interest.For example, when appropriately high titers of antibody to the immunogenare obtained, usually after repeated immunizations, blood is collectedfrom the animal and antisera are prepared. Further fractionation of theantisera to enrich for antibodies reactive to the protein can beperformed, if desired. See, e.g., Harlow and Lane Antibodies, ALaboratory Manual; or Coligan (ed.) Current Protocols in Immunology.Immunization can also be performed through other methods, e.g., DNAvector immunization. See, e.g., Wang, et al. (1997) Viroloqy228:278-284. Affinity purification, or absorptions, can be used toselect for desired specificity of binding.

[0049] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Typically, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell. See, Kohler and Milstein (1976) Eur. J.Immunol. 6:511-519. Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods known in the art. See, e.g., Doyle, et al. (eds. 1994 andperiodic supplements) Cell and Tissue Culture: Laboratory Procedures,John Wiley and Sons, New York, NY. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according, e.g., to the generalprotocol outlined by Huse, et al. (1989) Science 246:1275-1281.

[0050] Antibodies or binding compositions, including binding fragmentsand single chain versions, against predetermined fragments of MIP-3α orCCR6 polypeptides can be raised by immunization of animals withconjugates of the fragments with carrier proteins as described above.Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies can be screened for binding to normal ordefective MIP-3α protein, or screened for capacity to block cell MIP-3αmediated chemoattraction or chemokinetic activity. These monoclonalantibodies will usually bind with at least a KD of about 1 mM, moreusually at least about 300 μM, typically at least about 10 μM, moretypically at least about 30 μM, preferably at least about 10 μM, andmore preferably at least about 3 μM or better.

[0051] In some instances, it is desirable to prepare monoclonalantibodies (mAbs) from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basicand Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane (1988) Antibodies:A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andparticularly in Kohler and Milstein (1975) Nature 256:495-497, whichdiscusses one method of generating monoclonal antibodies. Summarizedbriefly, this method involves injecting an animal with an immunogen. Theanimal is then sacrificed and cells taken from its spleen, which arethen fused with myeloma cells. The result is a hybrid cell or“hybridoma” that is capable of reproducing in vitro. The population ofhybridomas is then screened to isolate individual clones, each of whichsecrete a single antibody species to the immunogen. In this manner, theindividual antibody species obtained are the products of immortalizedand cloned single B cells from the immune animal generated in responseto a specific site recognized on the immunogenic substance.

[0052] Other suitable techniques involve selection of libraries ofantibodies in phage or similar vectors. See, e.g., Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. Also, recombinant immunoglobulins may beproduced, see, Cabilly, U.S. Pat. No. 4,816,567; and Queen, et al.(1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; or made in transgenicmice, see Mendez, et al. (1997) Nature Genetics 15:146-156.

[0053] Antibody binding compounds, including binding fragments, of thisinvention can have significant diagnostic or therapeutic value. They canbe useful as non-neutralizing binding compounds and can be coupled totoxins or radionuclides so that when the binding compound binds to theantigen, a cell expressing it, e.g., on its surface, is killed. Further,these binding compounds can be conjugated to drugs or other therapeuticagents, either directly or indirectly by means of a linker, and mayeffect drug targeting.

[0054] C. Other Molecules

[0055] Antibodies are merely one form of specific binding compositions.Other binding compositions, which will often have similar uses, includemolecules that bind with specificity to MIP-3α receptor, e.g., CCR6, ina binding partner-binding partner fashion, an antibody-antigeninteraction, or in a natural physiologically relevant protein-proteininteraction, either covalent or non-covalent, e.g., proteins whichspecifically associate with MIP-3α receptor protein. The molecule may bea polymer, or chemical reagent. A functional analog may be a proteinwith structural modifications, or may be a structurally unrelatedmolecule, e.g., which has a molecular shape which interacts with theappropriate binding determinants. Application of, e.g., SystematicEvolution of Ligand by Exponential Enrichment (SELEX) technology,methods are available to select specific binding constructs for desiredtargets. See, e.g., Colas, et al. (1996) Nature 380:548-550; Cohen, etal. (1998) Proc. Nat'l Acad. Sci. USA 95:14272-14277; Kolonin, et al.(1998) Proc. Nat'l Acad. Sci. USA 95:14266-14271; Famulok, et al. (1998)Curr. Opin. Chem. Biol. 2:320-327; and Eaton, et al. (1997) Bioorg. Med.Chem. 5:1087-1096.

[0056] Drug screening using antibodies or MIP-3α or fragments thereofcan be performed to identify compounds having binding affinity toMIP-3α, or can block or simulate the natural interaction with ligand.Subsequent biological assays can then be utilized to determine if thecompound has intrinsic blocking activity and is therefore an antagonist.Likewise, a compound having intrinsic stimulating activity can signal tothe cells via the MIP-3α pathway and is thus an agonist in that itsimulates the activity of a ligand. Mutein antagonists may be developedwhich maintain receptor binding but lack signaling.

[0057] Structural studies of the ligands will lead to design of newvariants, particularly analogs exhibiting agonist or antagonistproperties on the receptor. This can be combined with previouslydescribed screening methods to isolate muteins exhibiting desiredspectra of activities.

[0058] As receptor specific binding molecules are provided, alsoincluded are small molecules identified by screening procedures. Inparticular, it is well known in the art how to screen for smallmolecules which interfere, e.g., with ligand binding to the receptor,often by specific binding to the receptor and blocking of binding bynatural ligand. See, e.g., meetings on High Throughput Screening,International Business Communications, Southborough, Mass. 01772-1749.Such molecules may compete with natural ligands, and selectively bind tothe MIP-3α or CCR6

[0059] III. Immunoassays

[0060] Immunoassays are valuable in diagnosing a disease or disorderassociated with MIP-3α imbalance or pathology. Qualitative orquantitative measurement of a particular protein can be performed by avariety of immunoassay methods. For a review of immunological andimmunoassay procedures in general, see Stites and Terr (eds. 1991) Basicand Clinical Immunology (7th ed.). Moreover, the immunoassays of thepresent invention can be performed in many configurations, which arereviewed extensively in, e.g., Maggio (ed. 1980) Enzyme Immunoassay CRCPress, Boca Raton, Fla.; Tijan (1985) “Practice and Theory of EnzymeImmunoassays,” Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers B.V., Amsterdam; Harlow and LaneAntibodies: A Laboratory Manual, supra;.Chan (ed. 1987) Immunoassay: APractical Guide Academic Press, Orlando, Fla.; Price and Newman (eds.1991) Principles and Practice of Immunoassays Stockton Press, NY; andNgo (ed. 1988) Non-isotopic Immunoassays Plenum Press, NY.

[0061] In particular, the present invention provides various skinrelated diseases as conditions susceptible to analysis or diagnosis byevaluating MIP-3α and/or CCR6. For example, the likelihood of skinrejection in a graft situation would be evaluated by the numbers ortypes of MIP-3α or CCR6 bearing cells present. Prophylacticdownregulation may be useful to prevent the recruitment of dermal T orNK cells. Response to various skin tumors may be evaluated by thepresence or absence of MIP-3α and/or CCR6 bearing cells.

[0062] Immunoassays for measurement of MIP-3α proteins or peptides canbe performed by a variety of methods known to those skilled in the art.In brief, immunoassays to measure the protein can be either competitiveor noncompetitive binding assays. In competitive binding assays, thesample to be analyzed competes with a labeled analyte for specificbinding sites on a capture agent bound to a solid surface. Preferablythe capture agent is an antibody specifically reactive with MIP-3αproteins produced as described above. The concentration of labeledanalyte bound to the capture agent is inversely proportional to theamount of free analyte present in the sample.

[0063] In a competitive binding immunoassay, typically the MIP-3αprotein present in the sample competes with labeled protein for bindingto a specific binding agent, e.g., an antibody specifically reactivewith the MIP-3α protein. The binding agent may be bound to a solidsubstrate or surface to effect separation of bound labeled protein fromthe unbound labeled protein. Alternately, the competitive binding assaymay be conducted in liquid phase and a variety of techniques known inthe art may be used to separate the bound labeled protein from theunbound labeled protein. Following separation, the amount of boundlabeled protein is determined. The amount of protein present in thesample is inversely proportional to the amount of labeled proteinbinding.

[0064] Alternatively, a homogeneous immunoassay may be performed inwhich a separation step is not needed. In these immunoassays, the labelon the protein is altered by the binding of the protein to its specificbinding agent. This alteration in the labeled protein results in adecrease or increase in the signal emitted by label, so that measurementof the label at the end of the immunoassay allows for detection orquantitation of the protein.

[0065] MIP-3α proteins may also be determined by a variety ofnoncompetitive immunoassay methods. For example, a two-site, solid phasesandwich immunoassay may be used. In this type of assay, a binding agentfor the protein, e.g., an antibody, is attached to a solid support. Asecond protein binding agent, which may also be an antibody, and whichbinds the protein at a different site, is labeled. After binding at bothsites on the protein has occurred, the unbound labeled binding agent isremoved and the amount of labeled binding agent bound to the solid phaseis measured. The amount of labeled binding agent bound is directlyproportional to the amount of protein in the sample.

[0066] Western blot analysis can be used to determine the presence ofMIP-3α or CCR6 proteins in a sample. Electrophoresis is carried out, forexample, on a tissue sample suspected of containing the protein.Following electrophoresis to separate the proteins, and transfer of theproteins to a suitable solid support, e.g., a nitrocellulose filter, thesolid support is incubated with an antibody reactive with the protein.This antibody may be labeled, or alternatively may be detected bysubsequent incubation with a second labeled antibody that binds theprimary antibody.

[0067] The immunoassay formats described above may employ labeled assaycomponents. The label may be coupled directly or indirectly to thedesired component of the assay according to methods well known in theart. A wide variety of labels and methods may be used. Traditionally, aradioactive label incorporating ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P was used.Non-radioactive labels include ligands which bind to labeled antibodies,fluorophores, chemiluminescent agents, enzymes, and antibodies which canserve as specific binding pair members for a labeled ligand. The choiceof label depends on sensitivity required, ease of conjugation with thecompound, stability requirements, and available instrumentation. For areview of various labeling or signal producing systems which may beused, see U.S. Pat. No. 4,391,904.

[0068] Antibodies reactive with a particular protein can also bemeasured by a variety of immunoassay methods. Thus modifications of theabove procedures may be used to determine the amounts or affinities ofvarious MIP-3α or CCR6 antibodies or antibody preparations. For a reviewof immunological and immunoassay procedures applicable to themeasurement of antibodies by immunoassay techniques, see, e.g., Stitesand Terr (eds.) Basic and Clinical Immunology (7th ed.) supra; Maggio(ed.) Enzyme Immunoassay, supra; and Harlow and Lane Antibodies, ALaboratory Manual, supra.

[0069] Screens to evaluate the binding and activity of mAbs and bindingcompositions encompass a variety of methods. Binding can be assayed bydetectably labeling the antibody or binding composition as describedabove. Cells responsive to MIP-3α can be used to assay antibody orbinding composition.

[0070] To evaluate MIP-3α chemoattraction or chemokinetic ability,experimental animals, e.g., mice, are preferably used. Skin, e.g.,Langerhans, cell counts are made prior to and at various time pointsafter administration of a bolus of the candidate agonist or antagonist.Levels are analyzed in various samples, e.g., blood, serum, nasal orpulmonary lavages, or tissue biopsy staining. A successful depleting mAbor binding composition will significantly lower the level of CCR6bearing cells. Such may be at least about 10%, preferably at least about20%, 30%, 50%, 70%, or more.

[0071] Evaluation of antibodies can be performed in other animals, e.g.,humans using various methods. For example, blood samples are withdrawnfrom patients suffering from a skin related disease or disorder beforeand after treatment with a candidate mAb.

[0072] IV. Uses

[0073] The exquisite tissue-selective homing of lymphocytes has longbeen appreciated as central for the control of systemic immuneresponses. Recent advances in the field support a model in whichleukocyte homing is achieved by sequential engagement of differentiallyexpressed and independently regulated vascular and leukocyte adhesionmolecules, and signaling receptors and their ligands. Butcher and Picker(1996) Science 272:60-66. The observation that chemokines, a superfamilyof small secreted proteins with G protein-coupled receptors (Baggiolini(1998) Nature 392:565-568) can attract leukocytes led to the hypothesisthat chemokines provide key signals directing recruitment of Tlymphocyte subsets into lymphoid and extra-lymphoid immune effectorsites. The inflamed skin-specific expression of MIP-3α and OCR6 suggeststhat such skin-specific chemokines selectively attract functionalsubsets of lymphocytes into the skin.

[0074] As such, the present invention provides means to purify desiredskin cell subsets. The chemoattractive or chemokinetic effects on thosecells can be the basis of purification methods. Methods exist forselective migration and recovery of cells to or from the chemokine,e.g., through porous membrane, or to various locations in a culture.Other methods exist to selectively separate cells of particular shapesfrom others. Alternatively, labeling can be used to FACS sort cellswhich specifically bind the chemokine. Populations of substantiallyhomogeneous Langerhans or skin derived cells will have important utilityin research or therapeutic environments.

[0075] While MIP-3α is likely to have functional effects on CCR6 bearingsubsets of cells, e.g., T and B cells-and precursors, other cells whichmay also be responsive include dendritic cells or granulocytes, e.g.,neutrophils and/or eosinophils, or their precursors. Effects on variouscell types may be indirect, as well as direct. A statisticallysignificant change in the numbers of cells will typically be at leastabout 10%, preferably 20%, 30%, 50%, 70%, 90%, or more. Effects ofgreater than 100%, e.g., 130%, 150%, 2×, 3×, 5×, etc., will often bedesired. The effects may be specific in causing chemotaxis to specificpoints, or may be chemokinetic, in inducing general movement of cells,but not necessarily in a specific direction, e.g., of concentrationgradient.

[0076] The present invention will be useful in the treatment of medicalconditions or diseases associated with immunological conditions of theskin. See, e.g., Bos (ed. 1990) Skin Immune System CRC Press, BocaRaton, Fla.; Fitzpatrick, et al. (eds. 1993) Dermatology in GeneralMedicine (4th ed.) McGraw-Hill, NY; Rook, et al. (eds. 1998) Textbook ofDermatology Blackwell; Habifor and Habie (1995) Clinical Dermatology: AColor Guide to Diagnosis and Therapy Mosby; Grob (ed. 1997)Epidemiology, Causes and Prevention of Skin Diseases Blackwell; Frank,et al. (eds. 1995) Samter's Immunologic Diseases, 5th Ed., vols. I-II,Little, Brown and Co., Boston, Mass.; Coffman, et al (1989) Science245:308-310; and Frick, et al. (1988) J. Allergy Clin. Immunol.82:199-225. The agonists or antagonists described may be combined withother treatments of the medical conditions described herein, e.g., anantibiotic, antifungal, antiviral, immune suppressive therapeutic,immune adjuvant, analgesic, anti-inflammatory drug, growth factor,cytokine, vasodilator, or vasoconstrictor. See, e.g, the Physician'sDesk Reference, both prescription and non-prescription compendiums.

[0077] The CCR6 receptor appears to be preferentially expressed on CD4+memory T cells. Its ligand, MIP-3α, is an inflammatory chemokineexpressed by cellular constituents of the skin, whose expression isinducible after stimulation with T cell-derived proinflammatorymeidators such as IFN-γ and IL-17. Thus, CD4+ memory T cell mediatedskin conditions are therapeutic targets of the antagonists, e.g.,psoriasis, atopic dermatitis, contact dermatitis, SLE, and lichen ruberplanus.

[0078] Preferred combination therapies include the MIP-3α reagent withvarious anti-inflammatory agents, such as topical, transdermal, orsystemic steroids or corticosteroids. Systemic, topical, transdermal, orsystemic retinoid or retinoid-like compounds, or vitamin D analogs, maybe administered with the MIP-3α therapeutics. Alternatively, variousforms of UV light may be used in combination with these therapeutics,e.g., ultraviolet A, ultraviolet B, or narrow bands of UVB.

[0079] For example, the MIP-3α ligands would be expected to signalspecifically to the cell types expressing their receptor. Thus, it willbe possible to block signaling, e.g., to the T cell or B cell subsets,by reagents which block receptor signaling, e.g., antibodies to ligand,and small drug antagonists.

[0080] Standard immunological techniques are described, e.g., inHertzenberg, et al. (eds. 1996) Weir's Handbook of ExperimentalImmunology vols. 1-4, Blackwell Science; Coligan (1991) CurrentProtocols in Immunology Wiley/Greene, NY; and Methods in Enzymologyvolumes 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163.These will allow use of the reagents for purifying cell subpopulations,etc.

[0081] To prepare pharmaceutical or sterile compositions including,e.g., MIP-3α, the material is admixed with a pharmaceutically acceptablecarrier or excipient which is preferably inert. Preparation of suchpharmaceutical compositions is known in the art, see, e.g., Remington'sPharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, MackPublishing Company, Easton, Pa. (1984). Typically, therapeuticcompositions are sterile. Alternatively, MIP-3α antagonist compositionscan be prepared.

[0082] Agonists, e.g., natural ligand, or antagonists, e.g., antibodiesor binding compositions, are normally administered parenterally,preferably intravenously. Since such protein or peptide antagonists maybe immunogenic they are preferably administered slowly, either by aconventional IV administration set or from a subcutaneous depot, e.g. astaught by Tomasi, et al., U.S. Pat. No. 4,732,863. However, as a skintarget, the administration may be topical, transdermal, intradermal,subcutaneous, or even systemic.

[0083] When administered parenterally the therapeutics will beformulated in a unit dosage injectable form (solution, suspension,emulsion) in association with a pharmaceutically acceptable parenteralvehicle. Such vehicles are inherently nontoxic and nontherapeutic. Theantagonist may be administered in aqueous vehicles such as water,saline, or buffered vehicles with or without various additives and/ordiluting agents. Alternatively, a suspension, such as a zinc suspension,can be prepared to include the peptide. Such a suspension can be usefulfor subcutaneous (SQ), intradermal (ID), or intramuscular (IM)injection. The proportion of therapeutic entity and additive can bevaried over a broad range so long as both are present in effectiveamounts. The therapeutic is preferably formulated in purified formsubstantially free of aggregates, other proteins, endotoxins, and thelike, at concentrations of about 5 to 30 mg/ml, preferably 10 to 20mg/ml. Preferably, the endotoxin levels are less than 2.5 EU/ml. See,e.g., Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: ParenteralMedications 2d ed., Dekker, NY; Lieberman, et al. (eds. 1990)Pharmaceutical Dosage Forms: Tablets 2d ed., Dekker, NY; Lieberman, etal. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker,NY; Fodor, et al. (1991) Science 251:767-773; Coligan (ed.) CurrentProtocols in Immunology; Hood, et al. Immunology Benjamin/Cummings; Paul(ed. 1997) Fundamental Immunology 4th ed., Academic Press; Parce, et al.(1989) Science 246:243-247; Owicki, et al. (1990) Proc. Nat'l Acad. Sci.USA 87:4007-4011; and Blundell and Johnson (1976) ProteinCrystallography, Academic Press, New York. Local, e.g., topical ortransdermal, administration will often be particularly useful.

[0084] Selecting an administration regimen for a therapeutic agonist orantagonist depends on several factors, including the serum or tissueturnover rate of the therapeutic, the immunogenicity of the therapeutic,or the accessibility of the target cells. Preferably, an administrationregimen maximizes the amount of therapeutic delivered to the patientconsistent with an acceptable level of side effects. Accordingly, theamount of therapeutic delivered depends in part on the particularagonist or antagonist and the severity of the condition being treated.Guidance in selecting appropriate doses of antibodies is found in theliterature on therapeutic uses, e.g. Bach et al., chapter 22, inFerrone, et al. (eds. 1985) Handbook of Monoclonal Antibodies NogesPublications, Park Ridge, N.J.; and Russell, pgs. 303-357, and Smith etal., pgs. 365-389, in Haber, et al. (eds. 1977) Antibodies in HumanDiagnosis and Therapy Raven Press, New York, N.Y.

[0085] Determination of the appropriate dose is made by the clinician,e.g., using parameters or factors known in the art to affect treatmentor predicted to affect treatment. Generally, the dose begins with anamount somewhat less than the optimum dose and it is increased by smallincrements thereafter until the desired or optimum effect is achievedrelative to any negative side effects. Numbers of CCR6 bearing cells indefined samples might be important indicators of when an effective doseis reached. Preferably, an antibody or binding composition thereof thatwill be used is derived from the same species as the animal targeted fortreatment, thereby minimizing a humoral response to the reagent.

[0086] The total weekly dose ranges for antibodies or fragments thereof,which specifically bind to MIP-3a, range generally from about 1 ng, moregenerally from about 10 ng, typically from about 100 ng; more typicallyfrom about 1 μg, more typically from about 10 μg, preferably from about100 pg, and more preferably from about 1 mg per kilogram body weight.Although higher amounts may be more efficacious, the lower dosestypically will have fewer adverse effects. Generally the range will beless than 100 mg, preferably less than about 50 mg, and more preferablyless than about 25 mg per kilogram body weight.

[0087] The weekly dose ranges for antagonists, e.g., antibody, bindingfragments, range from about 10 μg, preferably at least about 50 μg, andmore preferably at least about 100 μg per kilogram of body weight.Generally, the range will be less than about 1000 μg, preferably lessthan about 500 μg, and more preferably less than about 100 μg perkilogram of body weight. Dosages are on a schedule which effects thedesired treatment and can be periodic over shorter or longer term. Ingeneral, ranges will -be from at least about 10 μg to about 50 mg,preferably about 100 μg to about 10 mg per kilogram body weight.

[0088] Other antagonists of the ligands, e.g., muteins, are alsocontemplated. Hourly dose ranges for muteins range from at least about10 μg, generally at least about 50 μg, typically at least about 100 μg,and preferably at least 500 μg per hour. Generally the dosage will beless than about 100 mg, typically less than about 30 mg, preferably lessthan about 10 mg, and more preferably less than about 6 mg per hour.General ranges will be from at least about 1 μg to about 1000 μg,preferably about 10 μg to about 500 μg per hour.

[0089] In particular contexts, e.g., transplant or skin grafts, mayinvolve the administration of the therapeutics in different forms. Forexample, in a skin graft, the tissue may be immersed in a sterile mediumcontaining the therapeutic resulting in a prophylactic effect on cellmigration soon after the graft is applied.

[0090] The present invention also provides for administration of MIP-3αantibodies or binding compositions in combination with known therapies,e.g., steroids, particularly glucocorticoids, which alleviate thesymptoms associated with excessive inflammatory responses. Daily dosagesfor glucocorticoids will range from at least about 1 mg, generally atleast about 2 mg, and preferably at least about 5 mg per day. Generally,the dosage will be less than about 100 mg, typically less than about 50mg, preferably less than about 20 mg, and more preferably at least about10 mg per day. In general, the ranges will be from at least about 1 mgto about 100 mg, preferably from about 2 mg to 50 mg per day.

[0091] The phrase “effective amount” means an amount sufficient toeffect a desired response, or to ameliorate a symptom or sign of theskin condition. Typical mammalian hosts will include mice, rats, cats,dogs, and primates, including humans. An effective amount for aparticular patient may vary depending on factors such as the conditionbeing treated, the overall health of the patient, the method, route, anddose of administration and the severity of side affects. Preferably, theeffect will result in a change in quantitation of at least about 10%,preferably at least 20%, 30%, 50%, 70%, or even 90% or more. When incombination, an effective amount is in ratio to a combination ofcomponents and the effect is not limited to individual components alone.

[0092] An effective amount of therapeutic will modulate the symptomstypically by at least about 10%; usually by at least about 20%;preferably at least about 30%; or more preferably at least about 50%.Alternatively, modulation of migration will mean that the migration ortrafficking of various cell types is affected. Such will result in,e.g., statistically significant and quantifiable changes in the numbersof cells being affected. This may be an increase or decrease in thenumbers of target cells being attracted within a time period or targetarea.

[0093] The present invention provides reagents which will find use intherapeutic applications as described elsewhere herein, e.g., in thegeneral description for treating disorders associated with skinconditions. See, e.g., Berkow (ed.) The Merck Manual of Diagnosis andTherapy, Merck & Co., Rahway, N.J.; Thorn, et al. Harrison's Principlesof Internal Medicine, McGraw-Hill, NY; Gilman, et al. (eds. 1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8thEd., Pergamon Press; Remington's Pharmaceutical Sciences, 17th ed.(1990), Mack Publishing Co., Easton, Penn; Langer (1990) Science249:1527-1533; and Merck Index, Merck & Co., Rahway, N.J.

[0094] Antibodies to MIP-3α proteins may be used for the identificationor sorting of cell populations expressing MIP-3α protein, e.g.,fibroblasts or Langerhans cells. Methods to sort such populations arewell known in the art, see, e.g., Melamed, et al. (1990) Flow Cytometryand Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) PracticalFlow Cytometry Liss, New York, N.Y.; and Robinson, et al. (1993)Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y.Populations of cells expressing the MIP-3α receptor, e.g., CCR6, canalso be purified, e.g., using magnetic beads as described, e.g., inBieva, et al. (1989) Exp. Hematol. 17:914-920; Hernebtub, et al. (1990)Bioconj. Chem. 1:411-418; Vaccaro (1990) Am. Biotechnol. Lab. 3:30.

[0095] Moreover, antisense nucleic acids may be used. For example,antisense polynucleotides against the ligand encoding nucleic acids mayfunction in a manner like ligand antagonists, and antisense against thereceptor may function like receptor antagonists. Thus, it may bepossible to block the signaling through the pathway with antisensenucleic acids. Conversely, nucleic acids for the receptor may serve asagonists, increasing the numbers of receptor on the cell, therebyincreasing cell sensitivity to ligand, and perhaps blocking the normalapoptotic signal described.

[0096] Using the assay methods described above, the antibodies orbinding compositions are useful in diagnosing diseases states whichresult in skin disorders. Antibodies raised against a MIP-3α or CCR6protein will also be useful to raise anti-idiotypic antibodies. Thesewill be useful in detecting or diagnosing various immunologicalconditions related to expression of the respective antigens.Combinations of these signals may be also pursued.

[0097] The broad scope of this invention is best understood withreference to the following examples, which are not intended to limit theinventions to the specific embodiments.

EXAMPLES

[0098] I. General Methods

[0099] Some of the standard methods are described or referenced, e.g.,in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Bioloqy, Greene/Wiley, New York; Innis, et al.(eds.)(1990) PCR Protocols: A Guide to Methods and Applications AcademicPress, N.Y. Methods for protein purification include such methods asammonium sulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification” in Methods in Enzymology, vol. 182, and other volumes inthis series; manufacturer's literature on use of protein purificationproducts, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond,Calif.; and Coligan, et al. (eds.) (1995 and periodic supplements)Current Protocols in Protein Science, John Wiley & Sons, New York, NY.Combination with recombinant techniques allow fusion to appropriatesegments, e.g., to a FLAG sequence or an equivalent which can be fusedvia a protease-removable sequence. See, e.g., Hochuli (1989) ChemischeIndustrie 12:69-70; Hochuli (1990) “Purification of Recombinant Proteinswith Metal Chelate Absorbent” in Setlow (ed.) Genetic Engineering,Principle and Methods 12:87-98, Plenum Press, N.Y.; and Crowe, et al.(1992) QIAexpress: The Hiqh Level Expression & Protein PurificationSystem QIAGEN, Inc., Chatsworth, Calif.

[0100] Standard immunological techniques are described, e.g., inHertzenberg, et al. (eds. 1996) Weir's Handbook of ExperimentalImmunology vols. 1-4, Blackwell Science; Coligan (1991) CurrentProtocols in Immunology Wiley/Greene, NY; and Methods in Enzvmologyvolumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163.

[0101] Lymphocyte migration assays are performed as previouslydescribed, e.g., in Bacon, et al. (1988) Br. J. Pharmacol. 95:966-974.Other trafficking assays are also available. See, e.g.,Quidling-Järbrink, et al. (1995) Eur. J. Immunol. 25:322-327; Koch, etal. (1994) J. Clinical Investigation 93:921-928; and Antony, et al.(1993). J. Immunol. 151:7216-7223.

[0102] Alternatively, an activation assay or attraction assay is used.An appropriate cell type is selected, e.g., hematopoietic cells, myeloid(macrophages, neutrophils, polymorphonuclear cells, etc.) or lymphoid (Tcell, B cell, or NK cells), neural cells (neurons, neuroglia,oligodendrocytes, astrocytes, etc.), or stem cells, e.g., progenitorcells which differentiate to other cell types, e.g., gut crypt cells andundifferentiated cell types.

[0103] Chemokines may also be assayed for activity in hemopoietic assaysas described, e.g., by H. Broxmeyer. See Bellido, et al. (1995) J.Clinical Investigation 95:2886-2895; and Jilka, et al. (1995) Expt'lHematology 23:500-506. They may be assayed for angiogenic activities asdescribed, e.g., by Streiter, et al. (1992) Am. J. Pathol.141:1279-1284. Or for a role in inflammation. See, e.g., Wakefield, etal. (1996) J. Surgical Res. 64:26-31.

[0104] Other assays will include those which have been demonstrated withother chemokines. See, e.g., Schall and Bacon (1994) Current Opinion inImmunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy &Immunol. 109:97-109. Ca2+ flux upon chemokine stimulation is measuredaccording to the published procedure described in Bacon, et al. (1995)J. Immunol. 154:3654-3666.

[0105] FACS analyses are described in Melamed, et al. (1990) FlowCytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988)Practical Flow Cytometry Liss, New York, N.Y.; and Robinson, et al.(1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y.

[0106] II. Cell Culture and Tissue Samples

[0107] Adult human primary cells including keratinocytes, melanocytes,and dermal fibroblasts are obtained from Clonetics and culturedaccording to the suppliers instructions. For cytokine treatment, cellsare cultured with 10 ng/ml hTNF-α plus 3 ng/ml hIL-1β (R&D Systems) inculture medium. Human T cells are purified from PBMCs using a T-cellenrichment column (R&D Systems) according to the manufacturersinstructions.

[0108] III. Isolation of Encoding Sequences

[0109] The human, mouse, or rat MIP-3α sequence is readily available.See Table 1 and GenBank. Appropriate PCR primers or hybridization probescan be selected.

[0110] Similarly, the human CCR6, or others, can be readily isolated.See Table 2 and GenBank.

[0111] IV. Distribution Analysis

[0112] For Southern blotting, 5 μg of each cDNA library is digested withthe appropriate restriction enzymes to release the insert, subjected togel electrophoresis, and transferred to Hybond-N⁺ membrane. For Northernblotting all RNAs are isolated using RNAzol B (TEL-TEST, Inc.) andanalyzed by electrophoresis on a 1% formaldehyde-agarose gel andtransferred to Hybond-N⁺ membrane. Northern and Southern blots arehybridized for 16 hr at 650 C with ³²P-labeled probes obtained byrandomly priming (Prime-it; Stratagene) the full length inserts frommouse or human MIP-3α or CCR6 clones. After hybridization, blots arewashed at high-stringency and exposed to film.

[0113] The MIP-3α was identified from a cDNA library made from humanmonocytes activated with LPS and IFN-γ, in the presence of anti-IL-10.See, Rossi, et al. (1997) J. Immunology 158:1033-1036. Message of thechemokine has also been detected in pancreatic islet cells, fetal lung,and hepatic HEPG2 cells, suggesting a physiological role in inflammationor medical conditions in such organs/tissues.

[0114] The gene is expressed in HL-60 (promyelocytic leukemia); S3 (HeLacell); K562 (chronic myelogenous leukemia); MOLT-4 (lymphblasticleukemia); Raji (Burkitt's lymphoma); SW480 (colorectal adenocarcinoma);A549 (lung carcinoma); and G361 (melanoma) cell lines, as determined byprobing on a tissue blot from CLONTECH. Tissue expression gave apositive signal in lymph node, appendix, peripheral blood lymphocytes,fetal liver, and fetal lung, suggesting a physiological role ininflammation or medical conditions in such organs/tissues; but nodetectable signal in spleen, bone marrow, brain, and kidney.

[0115] The main transcript appears to be about 1.2 kb, with twoadditional transcript sizes in fetal lung RNA. Among the varioustissues, transcript sizes of 1.8, 2.7, and 4.2 kb were detected.

[0116] Positive signals were also detected in the following cDNAlibraries: dendritic cells activated with LPS, but not when activatedwith GM-CSF and IL-4; monocytes treated with LPS, IFN-γ, and anti-IL-10,but not when treated with LPS, IFN-γ, and IL-10; and activated PBMC.

[0117] These expression data implicate this chemokine in inflammatoryresponses upon cell activation. The lymph nodes, appendix, and PBL aresites where inflammatory processes take place. The MIP-3α may exert itseffects on monocytes and cells involved in inflammatory events. Otherstructural features implicate this chemokine in eosinophil and lungphysiology, e.g., asthma indications. Thus, an antagonist of thechemokine, e.g., an antibody, may be important for treatment ofasthmatic conditions. Also, IL-10 appears to inhibit MIP-3α expression.

[0118] The human MIP-3α is a ligand for the CCR6. Thus, a positivecontrol exists for the Ca++ flux assay for that receptor. This allowsfor the further screening of agonist ligands for the CCR6. Moreover, theCCR6 was isolated from eosinophil cDNA, and observations have been madethat eosinophils migrate to MIP-3α in vitro. See, e.g., Greaves, et al.(1997) J. Exp. Med. 186:837-844; Liao, et al. (1997) Biochem. Biophys.Res. Commun. 236:212-217; and Liao, et al. (1998) J. Immunol.162:186-194. These suggest that the MIP-3α interaction with the CCR6 isimportant in recruitment of eosinophils, as occurs with the eotaxinligand and the CCR3. As such, antagonists of the MIP-3α interaction withthe CCR6 will likely be useful in inhibiting eosinophilia, particularlyin the lung, or lung inflammation. These may accompany asthmatic orother pulmonary conditions. The specific upregulation of the pair ininflamed skin suggests a role in skin immunity.

[0119] The CCR6 was isolated from a cDNA library made from a dendriticcell cDNA library. It appears to be expressed in certain T cells, spleencell subsets, NK cells, and other cell populations enriched in dendriticcells, including CD1a⁺, CD14⁺, and CD1Aa⁺ cells. It did not give adetectable signal in TF1, Jurkat, MRC5, JY, or U937 cell lines.

[0120] Quantitative PCR methods have been applied, e.g., TAQMAN™ . Highlevels of CCR6 cDNA was detected in libraries made from peripheral bloodmononuclear cells, resting; T cell, THO clone Mot 72, resting; T cell,TH1 clone HY06, anergic; T cell clones, pooled, resting; T cell γδclones, resting; Splenocytes, resting; Splenocytes, activated; B cellEBV lines, resting; NK 20 clones pooled, resting; NK cell clone, NKA6;NK cytotoxic clone, resting; NK cell clone, NK non cytotox; monocytes,LPS, γIFN, anti-IL-10, 4+16 hr; monocytes, LPS, γIFN, IL-10, 4+16 hr; DC70% CD1a+, ex CD34+ GM-CSF, TNFα, activated 1 hr; DC 70% CD1a+, ex CD34+GM-CSF, TNFα, activated 6 hr; DC 95% CD1a+, ex CD34+ GM-CSF, TNFα,activated 1+6 hr; DC 95% CD14+, ex CD34+ GM-CSF, TNFα, activated 1+6 hr;DC CD1a+ CD86+, ex CD34+ GM-CSF, TNFα, activated 1+6 hr; DC resting CD34derived; DC CD4lo activated mo derived; DC resting activated mo derived;DC TGF and TGFb CD34 derived; lung fetal; gall bladder fetal; smallintestine fetal; ovary fetal; spleen fetal; normal human colon; normalhuman thyroid; tonsil inflammed; pool of three heavy smoker human lungsamples; allergic lung sample; Hashimoto's thyroiditis thyroid sample;and Psoriasis patient skin sample. Intermediate levels were detected inlibraries derived from peripheral blood mononuclear cells, activated; Tcell, THO clone Mot 72, activated; T cell, THO clone Mot 81, resting; Tcell, THO clone Mot 81, Activated; T cell, TH1 clone HY06, resting; Tcell, TH1 clone HY06, activated; B cell line JY, activated; NK 20 clonespooled, activated; NK cell clone, NKB1, pSPORT; NK cell clone, NKB1; DCex monocytes GM-CSF, IL-4, resting; DC ex monocytes GM-CSF, IL-4,monokine activated 4+16 hr; eosinophils; testes fetal; placenta 28 wk;pool of two normal human lung samples; ulcerative colitis human colonsample; pool of rheumatoid arthritis samples, human; and normal w.t.monkey colon. Low or undetectable levels were detected in libraries fromT cell, THO clone Mot 72, anergic; T cell, TH2 clone HY935, resting; Tcell, TH2 clone HY935, activated; T cell, TH1 clone TA20-23, resting; Tcell, TH1 clone TA20-23, activated; T cell, THO clone B21, resting; Tcell, THO clone B21, Activated; T cells CD4+, TH2 polarized, activated;T cell lines Jurkat and Hut78, resting; U937 premonocytic line, resting;U937 premonocytic line, activated; monocytes, LPS, γIFN, anti-IL-10;monocytes, LPS, γIFN, IL-10; monocytes, LPS, 1 hr; monocytes, LPS, 6 hr;DC 70% CD1a+, ex CD34+ GM-CSF, TNFα, resting; DC ex monocytes GM-CSF,IL-4, resting; DC ex monocytes GM-CSF, IL-4, LPS activated 4+16 hr;kidney fetal; liver fetal; heart fetal; brain fetal; Allergic lung #19;adipose tissue fetal; uterus fetal; normal human skin; Pneumocysticcarnii pneumonia lung sample; normal w.t. monkey lung;Ascaris-challenged monkey lung, 24 hr.; and Ascaris-challenged monkeylung, 4 hr.

[0121] Being found on dendritic cells, its ligand, including the MIP-3α,may be important in attracting appropriate cells for the initiation ofan immune response. MIP-3α has been shown to be a very potentchemoattractant for dendritic cells. Significant roles of the ligand andreceptor in skin physiology are suggested. The receptor may be alsopresent in other cells important in such responses.

[0122] V. Chemotaxis.

[0123] Recombinant mouse MIP-3α is produced in E. coli and purified,e.g., as previously described for other chemokines. Hedrick, et al.(1998) Blood 91:4242-4-247. Total human T cells in DMEM, pH 6.9, 1%bovine serum albumin, were added to the top chamber of 3 pm porepolycarbonate Transwell culture insert (Costar) and incubated with theindicated concentrations of purified chemokine in the bottom chamber for3 h. The number of migrating cells of each subtype is determined bymulti-parameter flow cytometry using fluorochrome conjugated antibodies.A known number of 15 μm microsphere beads (Bangs Laboratories, Fishers,Ind.) is added to each sample before analysis in order to determine theabsolute number of migrating cells.

[0124] Chemotaxis assays are performed with purified humanperipheral-blood T cells and/or skin-homing T cells. Other cell typesexpress the CCR6, e.g., T cells, B cells, DC cells, and granulocytecells, e.g., neutrophils and/or eosinophils. Recombinant murine MIP-3αshould have effects on the cell types expressing CCR6.

[0125] The MIP-3α and CCR6 expression levels are very low in normal skinsamples, but are highly upregulated in inflamed skin tissues.

[0126] VI. Antibody Production Appropriate mammals are immunized withappropriate amounts, e.g., of MIP-3α or MIP-3α gene transfected cells,e.g., intraperitoneally every 2 weeks for 8 weeks. Similar methods maybe used to produce antibodies which bind to CCR6, e.g., purified CCR6,polypeptides, or transfected cells expressing the receptor may be used.Typically, rodents are used, though other species should accommodateproduction of selective and specific antibodies. The final immunizationis given intravenously (IV) through the tail vein.

[0127] Generic polyclonal antibodies may be collected. Alternatively,monoclonal antibodies can be produced. For example, four days after theIV injection, the spleen is removed and fused to SP2/0 and NS1 cells.HAT resistant hybridomas are selected, e.g., using a protocol designedby Stem Cell Technologies (Vancouver, BC). After 10 days of HATselection, resistant foci are transferred to 96 well plates and expandedfor 3 days. Antibody containing supernatants are analyzed, e.g., by FACSfor binding to NIH3T3/surface MIP-3α transfectantsM Many differentMIP-3α mAbs are typically produced. Those antibodies may be isolated andmodified, e.g., by labeling or other means as is standard in the art.See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual CSHPress; Goding (1986) Monoclonal Antibodies: Principles and Practice (2ded.) Academic Press, New York, N.Y. Methods to conjugate magneticreagents, toxic entities, labels, attach the antibodies to solidsubstrates, to sterile filter, etc., are known in the art.

[0128] VII. Purification of Cells

[0129] MIP-3α responsive cells may be identified using the reagentsdescribed herein. For example, cells which are chemoattracted towardsMIP-3α may be purified from other cells by collecting those cells whichtraverse towards MIP-3α. Such chemotaxis may be to a source ofchemokine, or may be across a porous membrane or other substrate. Seeabove, in the microchemotaxis assay.

[0130] Alternatively, responsive cells may be identified by expressionof the receptor, e.g., CCR6, as provided herein. Thus, antibodies whichrecognize CCR6 may be used as a positive marker for sorting cells likelyto respond to MIP-3α. Conversely, the marker may be used to deplete CCR6bearing cells, e.g., by magnetic depletion or toxic conjugates.

[0131] Analysis of human samples can be evaluated in a similar manner. Abiological sample, e.g., blood, tissue biopsy sample, lung or nasallavage, skin punch, is obtained from an individual suffering from a skinrelated disorder. MIP-3α responsive cell analysis is performed, e.g., byFACS analysis, or similar means.

[0132] VIII. MIP-3α Antagonists

[0133] Various antagonists of MIP-3α are made available. For example,antibodies against the chemokine itself may block the binding of ligandto its receptor, thereby serving as a direct receptor antagonist. Otherantagonists may function by blocking the binding of ligand to receptor,e.g., by binding to the receptor in a way to preclude the possibility ofbinding of ligand. Other antagonists, e.g., mutein antagonists oraptamers, may bind to the receptor without signaling, thereby blocking atrue agonist from binding. Many of these may serve to block the signaltransmitted to target cells, specifically MIP-3α-responsive cells. Thesemay be skin cells, including Langerhans, fibroblasts, or keratinocytes.

[0134] Information on the criticality of particular residues isdetermined using standard procedures and analysis. Standard mutagenesisanalysis is performed, e.g., by generating many different variants atdetermined positions, e.g., at the positions identified above, andevaluating biological activities of the variants. This may be performedto the extent of determining positions which modify activity, or tofocus on specific positions to determine the residues which can besubstituted to either retain, block, or modulate biological activity.

[0135] Alternatively, analysis of natural variants can indicate whatpositions tolerate natural mutations. This may result from populationalanalysis of variation among individuals, or across strains or species.Samples from selected individuals are analyzed, e.g., by PCR analysisand sequencing. This allows evaluation of population polymorphisms.

[0136] IX. Psoriasis Studies

[0137] Psoriasis is a common chronic inflammatory skin diseasecharacterized by a marked inflammatory infiltrate and hyperproliferationof keratinocytes. The infiltrate is composed of skin-infiltrating Tcells, predominantly of the memory phenotype, neutrophils, liningmacrophages and increased numbers of dendritic cells. Ortonne (1996) Br.J. Dermatol. 135:Suppl 49:1-5; Prens, et al. (1995) Clin. Dermatol.13:115-129; and Elder, et al. (1994) J. Invest. Dermatol. 102:24S-27S.There is evidence that T cells play a crucial role in theimmunopathogenesis of this disease. See, e.g., Gottlieb, et al. (1995)Nature Med. 1:442-447; Nicolas, et al. (1991) Lancet 338:321; Cooper, etal. (1990) J. Am. Acad. Dermatol. 23:1318-1326; Ellis, et al. (1986)JAMA 256:3110-3116; Krueger, et al. (1995) J. Exp. Med. 182:2057-2068;Schon, et al. (1997) Nature Med. 3:183-188; Schon (1999) J. Invest.Dermatol. 112:405-410; Wrone-Smith and Nickoloff (1996) [see comments]J. Clin. Invest. 98:1878-1887; Uyemura, et al. (1993) J. Invest.Dermatol. 101:701-705; (1996) Arch. Dermatol. 132:419-423; and Barker(1998) Hosp. Med. 59:530-533. An early cellular event in the developmentof psoriatic lesions is the infiltration of target sites by activated Tcells which in turn produce inflammatory mediators, such as IFN-α,induce epidermal hyperplasia and may act with keratinocytes and dermalmacrophages to sustain a cycle of inflammation which finally leads to apsoriatic phenotype. Bata-Csorgo, et al. (1995) J. Invest. Dermatol.105:89S-94S.

[0138] A CC chemokine designated macrophage inflammatory protein-3α(MIP-3α; also known as LARC) was previously cloned and characterized,and CCR6 identified as its specific receptor. See, e.g., Rossi, et al.(1997) J. Immunol. 158:1033-1036; and Greaves, et al. (1997) J. Exp.Med. 186:837-844. MIP-3α is known to attract both T and dendritic cells.See Dieu, et al. (1998) J. Exp. Med. 188:373-386; and Power, et al.(1997) J. Exp. Med. 186:825-835. Among dendritic cells, MIP-3α is ahighly potent chemokine for the chemoattraction of epithelial Langerhanstype dendritic cells derived from CD34+ hematopoetic progenitor cells.Dieu, et al. (1998) J. Exp. Med. 188:373-386. Recently, MIP-3α has beenshown to preferentially attract the memory subset of T cells. Liao, etal. (1999) J. Immunol. 162:186-194; and Campbell, et al. (1998) Science279:381-384.

[0139] This study sought to identify chemokines and chemokine receptorsinvolved in autoimmune diseases. To this end, an analysis was made ofthe expression of mRNA of various chemokines and receptors in samples ofinflammatory skin diseases using real time quantitative PCR. Here isreported that the expression of the CC chemokine MIP-3α and its receptorCCR6 are significantly upregulated in psoriasis. Within psoriaticlesions, MIP-3α-expressing keratinocytes co-localize withskin-infiltrating T lymphocytes. Furthermore, CCR6 is expressed at highlevels on the skin-homing CLA+ subset of memory T cells. Finally,biologically active MIP-3α is induced in cellular constituents of theskin by proinflammatory cytokines and T cell-derived inflammatorymediators. Taken together, these observations strongly suggest that thisligand/receptor pair may represent a unique role in the development ofpsoriasis.

[0140] Patients: 6 mm punch biopsies were taken, after obtaininginformed consent, from either lesional (n=10) and non-lesional (n=5)skin of psoriatic patients or from normal (n=5) healthy individuals.Skin samples were immediately frozen in liquid nitrogen and stored at−80° C. In addition, 50 ml heparinized blood was drawn from eitherpsoriatic patients (n=15) in lesional phases of the disease or healthydonors (n=3) and peripheral blood mononuclear cells were prepared usingstandard protocols. The psoriatic patients used in this study had beenuntreated for at least three weeks.

[0141] Real Time Quantitative PCR (TaqMan®) Analysis of MIP-3α and CCR6mRNA Expression: Skin biopsies were homogenized in liquid nitrogen usinga Mikro-Dismembrator U (Braun Biotech, San Diego, Calif.) and RNA wasextracted with RNAzol according to the manufacturer's protocol(Tel-Test, Friedensburg, Tex.). 4 μg of RNA were treated with DNase I(Boehringer, Mannheim, Germany) and reverse transcribed with oligodT₁₄₋₁₈ (Gibco BRL, Gaithersburg, Md.) and random hexamer primers(Promega, Madison, Wis.) using standard protocols. cDNA was diluted to afinal concentration of 5 ng/μl. 10 μl of cDNA were amplified in thepresence of 12.5 μl of TaqMan® universal master mix (Perkin Elmer,Foster City, Calif.), 0.625 μl of gene-specific TaqMan® probe, 0.5 μl ofgene-specific forward and reverse primers, and 0.5 μl of water. As aninternal positive control, 0.125 μl of 18S RNA-specific TaqMan® probeand 0.125 μl of 18S RNA-specific forward and reverse primers were addedto each reaction. Specific primers and probes for MIP-3α, CCR6, and theother chemokine receptors measured were obtained from Perkin Elmer(Foster City, Calif.). Gene-specific probes used FAM as reporter whereasprobes for the internal positive control (18S RNA) were associated witheither the JOE or VIC reporters. Samples underwent the following stages:stage 1, 50° C. for 2 minutes; stage 2, 95° C. for 10 minutes; and stage3, 95° C. for 15 seconds followed by 60° C. for 1 minute. Stage 3 wasrepeated 40 times. Gene-specific PCR products were measured by means ofan ABI PRISM® 7700 Sequence Detection System (Perkin Elmer, Foster City,Calif.) continuously during 40 cycles. Specificity of primer probecombination was confirmed in crossreactivity studies performed againstplasmids of all known chemokine receptors (CCR1-CCR9, CXCR1-CXCR5, XCR1,CX3CR1) and the following panel of chemokines: MIP-1α, MIP-1β, MIP-1δ,MIP-3β, 6Ckine, IP-10, Mig, TCA-3, I-309, I-TAC, HCC-1, HCC-4, Gro-α/β,ENA78, eotaxin, eotaxin-2, DC-CK1, BCA-1, fractalkine, SDF-1α, RANTES,PF4, PBP, MDC, lymphotactin, IL-8, TARC, MDC, TECK, and MCP-1-MCP-4.Target gene expression was normalized between different samples based onthe values of the expression of the internal positive control. HumancDNA libraries used in this study were generated as describedpreviously. See, e.g., Rossi, et al. (1997) J. Immunol. 158:1033-1036;Bolin, et al. (1997) J. Neurosci. 17:5493-5502; and Vicari, et al.(1997) Immunity 7:291-301.

[0142] Cell Culture: Human primary epidermal keratinocytes, dermalfibroblasts, melanocytes, and dermal microvascular endothelial cellswere purchased from Clonetics (San Diego, Calif.) and cultured inkeratinocyte (KGM), fibroblast (FGM), melanocyte (MGM), or endothelialcell (EGM-2) growth medium (Clonetics, San Diego, Calif.) as describedin Detmar, et al. (1994) J. Exp. Med. 180:1141-1146. Cells were treatedwith TNF-α (10 ng/ml) / IL-1β (5 ng/ml), IFN-γ (20 ng/ml), IL-4 (50ng/ml), IL-17 (100 ng/ml) (R&D Systems Inc., Mineapolis, Minn.), or leftuntreated. The epidermal γδ T cell line, 7-17, was kindly provided byRichard Boismenu (The Scripps Institute, La Jolla, Calif.) and culturedas described previously. See Boismenu, et al. (1996) J. Immunol.157:985-992. Epidermal γδ T cells were cultured with Con A, TNF-α (10ng/ml) / IL-1γ (5 ng/ml), or medium alone. Supernatants as well as cellswere harvested after 6 or 18 h. Generation of dendritic cells eitherfrom cord blood CD34+ hematopoetic progenitor cells or from peripheralblood monocytes was performed as described in Dieu, et al. (1998) J.Exp. Med. 188:373-386. Immature dendritic cells from CD34+ hematopoeticprogenitor cells or monocyte-derived dendritic cells were activated for3-24 h in the presence of CD40L transfected L cells (one per fivedendritic cells) as described. See Caux, et al. (1994) J. Exp. Med.180:1263-1272. Cells and supernatants were harvested 3, 12, 24, 48 hoursafter CD40L stimulation. RNA was extracted from cells as describedabove.

[0143] Flow Cytometry and Cell Sorting: In order to analyze chemokinereceptor expression of skin-homing T cell subsets, CLA⁺/CD45RO⁺/CD4⁺cells were sorted from PBMCs isolated from 2 different donor pool buffycoats (70 ml) from 3 individual donors each using a FACS Vantage (BectonDickinson, San Jose, Calif.) and the following monoclonal antibodies(mAb) (Pharmingen, San Diego, Calif.): FITC-conjugated anti-CLA(HECA4522) mAb, PE-conjugated anti-CD45RO (UCHL1), APC-conjugatedanti-CD4 (RPA-T4). The purity of the cells was detected as >99.5%.Subsequently, RNA was extracted and reverse transcribed as describedabove.

[0144] In other experiments, CCR6 expression was analyzed on memory Tcell subsets using the following antibodies: FITC-conjugated anti-CLA(HECA4522) mAb (Pharmingen, San Diego, Calif.), FITC-conjugatedanti-CD45RO (UCHL1) mAb (Pharmingen, San Diego, Calif.), APC-conjugatedafiti-CD8 (RPA-T8) mAb (Pharmingen, San Diego, Calif.),Cy-chrome-conjugated anti-CD4 (RPA-T4) mAb (Pharmingen, San Diego,Calif.), anti-CCR6-PE conjugated (53103.111) mAb (R&D Systems,Mineapolis, Minn.), mouse IgG_(2b)-PE-conjugated (R&D Systems,Mineapolis, Minn.). Briefly, 10⁶ PBMCs were stained with anti-CD4,anti-CD8, anti-CLA, anti-CCR6 mAb or isotype and analyzed using aFACSCalibur and CELLQuest software (Becton Dickinson, San Jose, Calif.).

[0145] In Situ Hybridization: In situ hybridization was performed asdescribed. Dieu, et al. (1998) J. Exp. Med. 188:373-386. Coupled primerswere used for amplifying by RT-PCR the majority of the open readingframe of the MIP-3α gene. +77/MIP-3α forward primer and −425/MIP-3α wereused with an annealing temperature of 62° C. Then, PCR products werecloned into pCRII TA cloning vector (Invitogen, Leek, The Netherlands),RNA probes were synthesized using Sp6 and T7 RNA polymerases (BoehringerMannheim, Germany) and radiolabled with ³⁵S-UTP (Amersham Corp., UnitedKingdom). Sense and anti-sense ³⁵S-labeled probes of MIP-3α wereobtained by run-off transcription of the 367 bp fragment and thenpartially degraded by alkaline hydrolysis for 20 min at 60° C. 6 μmcryostat sections were prepared on charged electrostatic slides(SuperFrost/Plus, Polylabo, Strasbourg, France) and fixed with coldacetone for 20 min, with 4% paraformaldehyde for 20 min followed by 0.1M triethanolamine/0.25% acetic anhydride. The sections were hybridizedovernight at 50° C. (2 to 3×10⁶ cpm/slide), RNAse A treated, and exposedfor 40 days. After development, the sections were stained withhematoxylin.

[0146] Immunohistochemistry: Frozen 6 μm tissue sections were fixed inacetone and in paraformaldehyde before the immunostaining. To block thenon-specific binding.of avidin, biotin system components, or endogenousperoxidase activity, sections were pre-treated with avidin D and biotinsolutions (Blocking kit, Vector, Biosys SA, Compiegne, France) for 10min each step and with PBS containing 0.3% hydrogen peroxide (Sigma,France) for 15 min at room temperature. After brief washing in PBS, thesections were incubated with blocking serum (2% normal rabbit serum) forat least 30 min before adding both primary antibodies. Sections weredouble stained simultanously with anti-hMIP-3α goat polyclonal antibody(IgG isotype, R&D Systems Inc., Minesota, Minn.) and anti-hCD3 mousemonoclonal antibody (Leu-4, IgG1 isotype, Becton-Dickinson, MoutainView, Calif.) for 1 hour at room temperature in a humid atmosphere. Thebinding of goat IgG was detected using biotinylated rabbit anti-goat IgGfollowed by streptavidin-peroxidase (both included in the Vectastain ABCkit: Goat IgG PK-4005, Vector) and the binding of mouse IgGl wasdetected by rabbit alkaline phosphatase-labeled anti-mouse IgG (D0314,Dako, Glostrup, Denmark) at the same time at room temperature in a humidatmosphere. The peroxidase and alkaline phosphatase activities wererevealed using 3-amino-9-ethylcarbazole (AEC) substrate (SK-4200,Vector) and alkaline phosphatase substrate III (SK-5300, Vector) for 5to 10 min at room temperature, respectively. Negative controls wereestablished by adding non-specific isotype controls as primaryantibodies.

[0147] Generation of Mouse mAbs Against hMIP-3α and Development of anhMIP-3α ELISA: Inbred BALB/c mice were immunized with three successiveintraperitoneal injections of complete Freund's adjuvant (Sigma ChemicalCo., St. Louis, Mo.), incomplete Freund's adjuvant, or without Freund'sadjuvant, respectively, with 50 ng of purified hMIP-3α obtained fromsupernatants of hMIP-3α transient transfected COP5 cells. Spleens wereremoved for fusion 3 days after a final i.v. injection of hMIP-3α.Hybridization was carried out using the non-secreting myeloma cell lineSP2/0-Ag8 with polyethylene glycol 1000 (Sigma Chemical Co, St. Louis,Mo.). hMIP-3α transient transfected COP5 cells were cultured for 2 daysin 96 well plates and fixed in acetone. Then, hybridoma supernatantswere harvested after 6 days, were incubated for 30 min on fixed hMIP-3αtransient transfected COP5 cells. Antibody binding was then revealedwith peroxidase-conjugated sheep anti-mouse IgG (Biosys, Compiegne,France) at a 1:200 dilution in PBS for 30 min at 37° C. Positivehybridomas were cloned by limiting dilution and expanded using a highdensity culture system (Integra cell line CL1000, Integra Biosciences,France). After sodium sulphate precipitation, the mAbs were purified byanion-exchange chromatography on a Hyper-D column and peroxidase labeled(Sepracor, Villeneuve, France). An ELISA was set up using one of theanti-hMIP-3α mAbs, 319F6, as a capture mAb and a peroxidase-coupledmouse anti-hMIP-3α mAb to reveal the captured hMIP-3α. The assay provedto be specific for MIP-3α with a sensitivity of 0.2 ng/ml.

[0148] Analysis of hMIP-3α bioactivity by calcium mobilization assay: Acell line expressing the human CCR6 chemokine receptor was kindlyprovided by Chuan Chu Chou (SPRI, Kennilworth, N.J.). Briefly, the CCR6open reading frame was cloned into the pME18sneo eukaryotic expressionvector and transfected into the murine B-cell line, BAF/3 byelectroporation. Stable transfectants were isolated by selection inmedium containing 1 mg/ml G418. CCR6 expression was confirmed usingcalcium signaling, ligand binding analysis with recombinant human MIP-3α(R&D Systems, Minneapolis, Minn.), and immunohistochemistry withanti-CCR6 (53103.111) mAb (R&D Systems, Minneapolis, Minn.). The averagenumber of binding sites per cell was estimated to be 220,000.

[0149] To measure the biological activity of the MIP-3α produced bykeratinocytes fibroblast or endothelial cells, supernatants from thesecell cultures were concentrated 20-fold using Centriplus concentratorswith a cut off of 3 kD (Amicon, Beverly, Mass.). The calcium response tosupernatants from these resting or activated cells was measured usingstandard protocols. See, e.g., Greaves, et al. (1997) J. Exp. Med.186:837-844. Briefly, the BAF/3 parental and CCR6 transfectant wereloaded for 60 min at 37° C. with 3 μM INDO-1A (Molecular-Probes, Eugene,Oreg.). Cells were washed and resuspended in Hank's balanced saltsolution (HBSS) (Gibco/BRL, Grand Island, N.Y.) to a final concentrationof 10⁷ cells/ml. Calcium mobilization was measured using a Photon,Technology International spectrophotometer with excitation at 350 nm anddual simultaneous recording of fluorescence emission at 400 nm and 490nm. Relative intracellular calcium levels are expressed as the 400nm/490 nm emission ratio. Experiments were performed at 37° C. withconstant mixing in cuvettes containing 10⁶ cells in 2 ml of HBSS with 1mM CaCl₂. In order to demonstrate specificity of MIP-3α-induced calciummobilization, neutralization studies were performed using a blockingmouse anti-human MIP-3α (IgG₁) (67310.111) mAb (R&D Sytems, Minneapolis,Mo.) or isotype control (IgG₁) (Sigma, St. Louis Mo.).

[0150] MIP-3α and its Specific Receptor CCR6 Are SignificantlyUpregulated in Lesional Psoriatic Versus Non-Lesional or Normal Skin:After cloning and initial characterization of mouse and human MIP-3α andidentification of its receptor, CCR6, the potential role of thisligand-receptor pair was investigated in human diseases. See Rossi, etal. (1997) J. Immunol. 158:1033-1036; and Greaves, et al. (1997) J. Exp.Med. 186:837-844. To this end, a systematic screening of human tissuecDNA libraries with MIP-3α- and CCR6-specific TaqMan® probe and primerswas undertaken. The cDNA library panel included various librariesderived from human autoimmune disease samples. This initial screeningshowed that MIP-3α was expressed more than 100-times higher in a cDNAlibrary derived from lesional psoriatic skin (568000 fg/50 ng cDNA) whencompared to normal skin (5530 fg/50 ng cDNA). Moreover, TaqMan® analysesshowed abundant CCR6 message (864 fg/50 ng cDNA) in the cDNA librarygenerated from psoriatic skin. In contrast, CCR6 was undetectable in acDNA library derived from normal skin. Other chemokine receptors thathave been reported to be upregulated in psoriasis were investigated inthe cDNA libraries from normal and psoriatic skin. To this end, theexpression of the IL-8 receptors was studied, and confirmed previousreports. See Kulke, et al. (1998) J. Invest. Dermatol. 110:90-94; andSchulz, et al. (1993) J. Immunol. 151:4399-4406. CXCR1 and CXCR2 wereconstitutively expressed in the cDNA library derived from normal skinand markedly upregulated in the psoriatic skin cDNA library. Thus, thecDNA libraries derived from normal or psoriatic skin providedrepresentative tools to study gene expression. These initialobservations prompted investigation of a possible role for MIP-3α andCCR6 in the pathogenesis of psoriasis in more detail. These led toefforts to validate these observations in more patient samples.Consecutive quantitative real time PCR analysis of cDNA derived fromeither lesional (n=10), non-lesional (n=5) psoriatic or normal (n=5)skin confirmed that both MIP-3α and CCR6 were significantly upregulatedin lesional psoriatic versus non-lesional or normal skin (P<0.005). Anaverage 7- and 4-fold induction of MIP-3α and CCR6 could be detected,respectively. To look more precisely at the distribution of MIP-3α, insitu hybridization and immunohistochemistry were performed usingspecific probes and polyclonal antibodies directed against MIP-3α. Insitu hybridization with a MIP-3α specific probe showed selectiveexpression of this chemokine in the suprabasal layers of the epidermisof lesional psoriatic skin. These showed specific hybridization withinthe stratum granulosum of lesional psoriatic skin. In addition, MIP-3αmRNA expression could be detected within the stratum spinosum ofpsoriatic epidermis. Sense (vs antisense) controls as well as in situhybridization with normal and non-lesional psoriatic skin confirmed thespecific detection of MIP-3α hybridization in lesional psoriatic skin.Furthermore, irmunohistochemical staining of lesional psoriatic skinconfirmed the focal upregulation of MIP-3α protein within suprabasallayers of the epidermis. In contrast, non-lesional psoriatic and normalskin showed no specific staining for MIP-3α. Staining with isotypecontrols showed the specificity of MIP-3α detection. Attempts to detectCCR6by immunohistochemistry for CCR6 were inconclusive due to the lowsensitivity of the antibody. Therefore, double stainings were performedfor MIP-3α and CD3 to study localization of MIP-3(-expressing cells andT cells. Immunohistochemisty of lesional psoriatic skin showed thatfocal accumulation of T cells in the papillary dermis of lesionalpsoriatic skin was directly adjacent to foci of MIP-3α-expressingepidermal cells. Moreover, MIP-3α-expressing keratinocytes within theepidermis co-localized with intraepidermal CD3⁺ T cells.

[0151] Peripheral Blood Mononuclear Cells From Psoriasis PatientsExpress Significantly Higher Levels of CCR6 When Compared With Those ofHealthy Donors: Clinically, it is well known that infections may triggerpsoriatic episodes and recently it has been suggested that superantigensmay play a role in T cell activation during the pathogenesis ofpsoriasis. See Valdimarsson, et al. (1997) Clin. Exp. Immunol. 107 Suppl1:21; Nickoloff, et al. (1993) J. Immunol. 150:2148-2159; and Nickoloff,et al. (1993) J. Dermatol. Sci. 6:127-133. However, very little is knownabout chemokine receptor expression on PBMCs of normal healthy donorsversus psoriatic patients. Interestingly, CCR6 was also significantlyupregulated in PBMCs derived from psoriasis patients (n=10) versus PBMCfrom healthy donors (n=5) (P<0.001). PBMCs from psoriatic patientsexpressed on average 4-fold higher levels of CCR6 mRNA when comparedwith PBMCs from healthy donors. The CLA+ T cell subset represents askin-associated population of memory T cells that migratespreferentially to normal and chronically inflamed cutaneous sites. SeePicker, et al. (1990) J. Immunol. 145:3247-3255.

[0152] Subsequent experiments focused on the chemokine receptor profileof pathologically relevant skin-homing CLA⁺ memory T cells. Flowcytometric analyses revealed that CCR6 was expressed at high levels onthe surface of skin-homing CLA⁺ T cells of normal donors. Moreover, CCR6was predominantly expressed on the CD4⁺ subset of CLA⁺ T cells. Thelatter observation may account for the therapeutic effect of anti-humanCD4 antibodies in the treatment of psoriasis. See Isaacs, et al. (1997)Clin. Exp. Immunol. 110:158-166; Thivolet and Nicolas (1994) Int. J.Dermatol. 33:327-332; and Morel, et al. (1992) J. Autoimmun. 5:465-477.

[0153] In agreement with flow cytometric analyses, TaqMan® analyses onsorted CLA+ memory T cells from normal donors indicated that theyexpress CCR6 at high levels. In fact, the expression of CCR6 was thehighest one detected among chemokine receptors, especially when comparedto receptors of other chemokines (IL-8, Gro-α, IP-10, Mig, MCP-1,RANTES) that have already been associated with psoriasis. See Schulz, etal. (1993) J. Immunol. 151:4399-4406; Gottlieb, et al. (1988) J. Exp.Med. 168:941-948; Lemster, et al. (1995) Clin. Exp. Immunol. 99:148-154;Gillitzer, et al. (1993) J. Invest. Dermatol. 101:127-131; Fukuoka, etal. (1998) Br. J. Dermatol. 138:63-70; and Goebeler, et al. (1998) J.Pathol. 184:89-95. CCR6 expression was 100 to more than 1000-timeshigher than CXCR1, CXCR2, CXCR3, CCR2, CCR3 and CCR5 on this skin-homingsubset of memory T cells.

[0154] TNF-α, IL-1β, IFN-γ, IL-17, and CD40L Regulate the Expression ofMIP-3α in Cellular Constituents of the Skin: The pattern of MIP-3αexpression within the epidermis suggested that keratinocytes may be amajor source for MIP-3α in the skin. To further investigate the cellularorigin of MIP-3α within the skin and to get insights into itsregulation, human primary keratinocytes, melanocytes, and dermalfibroblasts were cultured with TNF-α/IL-1β, IFN-γ, IL-4, IL-17, ormedium alone as control. Furthermore, it was tested whether culturedepidermal γδ T cells resting or stimulated with either TNF-α/IL-1β orCon A may express MIP-3α. Human primary dermal microvascular endothelialcells were also cultured in the presence or absence of TNF-α/IL-1β.TNF-α and IL-1β were used for stimulation since these proinflammatorycytokines are known to be upregulated during inflammatory conditions andin psoriatic lesional skin. See Terajima, et al. (1998) Arch. Dermatol.Res. 290:246-252; Mizutani, et al. (1997) J. Dermatol. Sci. 14:145-153;Debets, et al. (1995) Eur. J. Immunol. 25:1624-1630; Nickoloff, et al.(1991) Am. J. Pathol. 138:129-140; and Gomi, et al. (1991) [seecomments] Arch. Dermatol. 127:827-830. Furthermore, the effects of Thelper cell-derived cytokines such as IL-4, IFN-γ, and IL-17 were testedon cellular constituents of the skin. TaqMan® analyses showed that onlykeratinocytes and dermal microvascular endothelial cells constitutivelyexpress low levels of MIP-3α and that TNF-α/IL-1β can induce MIP-3α mRNAexpression in both keratinocytes and dermal microvascular endothelialcells. In addition, TNF-α/IL-1β stimulation of dermal microvascularendothelial cells induced strong upregulation of MIP-3α expression.Activation of both CD34+hematopoetic progenitor cell- ormonocyte-derived dendritic cells with CD40L also induced MIP-3α-specifictranscripts. Interestingly, melanocytes also showed significantexpression of MIP-3α following TNF-α/IL-1β stimulation. Moreover, MIP-3αexpression could be markedly induced in these cells by IFN-γ or IL-4stimulation. Keratinocytes showed a weak (2-4 fold) upregulation ofMIP-3α mRNA after activation with either IFN-γ or IL-4. In contrast,resting or stimulated epidermal γδ cells failed to express MIP-3α mRNAunder any conditions. Similar results were observed in keratinocytes(n=4), melanocytes (n=2), CD34+ hematopoietic progenitor cell-deriveddendritic cells (n=2), monocyte-derived dendritic cells (n=2), dermalmicrovascular endothelial cells (n=2) and dermal fibroblasts (n=2) ofdifferent donors. Interestingly, keratinocytes, fibroblasts,melanocytes, or epidermal γδ T cells were never observed to expresssignificant levels of CCR6 mRNA.

[0155] Analyses of MIP-3α protein expression by ELISA confirmed thatactivated keratinocytes, dermal microvascular endothelial cells, dermalfibroblasts, and monocyte-derived dendritic cells are potent producersof this chemokine. Supernatants from cultured primary humankeratinocytes, dermal microvascular endothelial cells, and dermalfibroblasts activated with TNF-α and IL-1β showed a marked induction ofMIP-3α protein while resting cells showed either little or no productionof MIP-3α. These levels of hMIP-3α production are in the range ofbiological activity reported for prokaryote-derived recombinant hMIP-3α.Low levels of MIP-3α protein were also detected after stimulation ofkeratinocytes with IFN-γ or IL-17, however, additional TNF-α stimulationshowed synergistic effects and markedly enhanced MIP-3α proteinproduction.

[0156] Supernatants of monocyte-derived dendritic cells showedsignificant production of MIP-3α protein (1.14-8.76 ng/ml) 12 to 48 hfollowing CD40L stimulation. MIP-3α protein expression generallyconfirmed the data obtained using real time quantitative PCR (TaqMan⁺).However, supernatants from CD34+ hematopoetic progenitor cellsstimulated with CD40L did not contain detectable MIP-3α protein despitea marked induction of MIP-3α mRNA expression. This may be due to thehigh level of CCR6 expression by these cells suggesting that they may bebinding and internalizing MIP-3α.

[0157] It was then sought to determine if the MIP-3α protein detected inthese supernatants was biologically active. To this end, supernatantswere tested against keratinocytes, fibrobasts, or endothelial cells,either resting or following stimulation with TNF-α/IL-1β, IFN-γ, IL-4,in a calcium signaling assay using CCR6-transfected BAF/3 cells. Thesecells are known to express endogenous CXCR4. Therefore, to obtain aCCR6-specific assay, the endogenous CXCR4 was blocked with human SDF-1αprior to testing the supernatants for MIP-3α activity. In agreement withthe ELISA data, supernatants from keratinocytes, dermal fibroblasts, anddermal microvascular endothelial cells stimulated with TNF-α/IL-1βinduced significant calcium mobilization responses in CCR6-transfectedBAF/3 cells but not in the parental untransfected BAF/3 cells. However,the parental BAF/3 cell line showed the expected calcium mobilizationresponse due to the triggering of endogenous CXCR4 by SDF-1α.Furthermore, treatment with anti-MIP-3α mAb completely neutralizedsupernatant-induced calcium mobilization in CCR6-transfected BAF/3cells, however, isotype control showed no effect. Concentrated mediumwith or without cytokine (TNF-α/IL-1β, IL-4, IFN-γ) addition did notproduce any intracellular Ca++ mobilization in parental orCCR6-transfected cells. These results confirm the production ofbioactive MIP-3α protein by keratinocytes, dermal fibroblasts, anddermal microvascular endothelial cells initially detected at the mRNAlevel by quantitative PCR.

[0158] This study demonstrates that the CC chemokine MIP-3α and itsreceptor CCR6 are significantly upregulated in psoriasis. Furthermore,clusters of skin-infiltrating T cells in the papillary dermis oflesional psoriatic skin are directly adjacent to foci ofMIP-3α-expressing epidermal cells. Moreover, MIP-3α-expressingkeratinocytes within the epidermis co-localize with intraepidermal CD3⁺T cells. These findings together with the observation of Liao, et al.(1999) J. Immunol. 162:186-194 that MIP-3α specifically attracts thememory subset of T cells in vitro strongly suggests that MIP-3α plays animportant role in T cell recruitment to lesional psoriatic skin. Thesignificantly increased expression of CCR6 in PBMCs derived frompsoriatic donors and the high expression of CCR6 on skin-homing CLA Tcells further supports this concept. In addition to memory T cells,peripheral blood B cells also express CCR6, however, they are notpresent in psoriatic skin lesions (Bata-Csorgo, et al. (1995) J. Invest.Dermatol. 105:89S-94S; Liao, et al. (1999) J. Immunol. 162:186-194; Bos,et al. (1983) Arch. Dermatol. Res. 275:181-189; and Bos, et al. (1989)Arch. Dermatol. Res. 281:24-30) suggesting that there may be furthernecessary requirements such as E-selectin or CLA expression foreffective skin homing. The CLA+ T cell subset represents askin-associated population of memory T cells that preferentiallyextravasates to normal and chronically inflamed cutaneous sites. SeePicker, et al. (1990) J. Immunol. 145:3247-3255. Comparison of CCR6expression in CLA+ T cells with those of receptors for chemokines (IL-8;Gro-α, IP-10, MIG, MCP-1, RANTES) reported to be associated withpsoriasis underscore the relevance of this specific chemokine/receptorpair in psoriasis. Schulz, et al. (1993) J. Immunol. 151:4399-4406;Gottlieb, et al. (1988) J. Exp. Med. 168:941-948; Lemster, et al. (1995)Clin. Exp. Immunol. 99:148-154; Gillitzer, et al. (1993) J. Invest.Dermatol. 101:127-131; Fukuoka, et al. (1998) Br. J. Dermatol.138:63-70; and Goebeler, et al. (1998) J. Pathol. 184:89-95.Furthermore, the predominance of CCR6 expression on the CD4 subset ofskin-homing CLA+ T cells suggests a link with the effective treatment ofpsoriasis using anti-human CD4 antibodies. See Isaacs, et al. (1997)Clin. Exp. Immunol. 110:158-166; Thivolet and Nicolas (1994) Int. J.Dermatol. 33:327-332; and Morel, et al. (1992) J. Autoimmun. 5:465-477.These findings support previous observations by Campbell, et al. (1998)Science 279:381-384 showing that MIP-3α induces rapid adhesion to ICAM-1only in memory but not in naive CD4+ T cells. Furthermore,immunohistological studies have shown that the inflammatory infiltratein psoriasis is mainly composed of CD4+ memory T cells. See Prens, etal. (1995) Clin. Dermatol. 13:115-129; and Bata-Csorgo, et al. (1995) J.Invest. Dermatol. 105:89S-94S.

[0159] Interestingly, TaqMan® analyses of cDNA libraries show that CCR6is predominantly expressed in cDNA libraries derived from human Th₀ orTh₁ clones. However, cDNA libraries derived from either activated Th₂polarized cells or resting or activated Th₂ (HY935) clones show littleor no CCR6 expression. This expression pattern supports previousobservations indicating that lesional psoriatic T cells predominantlydisplay a Th₁ phenotype. See Uyemura, et al. (1993) J. Invest. Dermatol.101:701-705; Barker (1998) Hosp. Med. 59:530-533; Nestle, et al. (1994)J. Clin. Invest. 94:202-209; and Schlaak, et al. (1994) J. Invest.Dermatol. 102:145-149. In addition, preliminary experiments show thatactivated T cells are capable of inducing high levels of MIP-3αproduction in epithelial cells.

[0160] Keratinocytes are potent producers of MIP-3α in lesionalpsoriatic skin. Here it is shown that TNF-α and IL-1, bothproinflammatory cytokines known to be upregulated in psoriasis, (seeTerajima, et al. (1998) Arch. Dermatol. Res. 290:246-252; Mizutani, etal. (1997) J. Dermatol. Sci. 14:145-153; Debets, et al. (1995) Eur. J.Immunol. 25:1624-1630; Nickoloff, et al. (1991) Am. J. Pathol.138:129-140; and Gomi, et al. (1991) [see comments] Arch. Dermatol.127:827-830) as well as CD40L are potent inducers of bioactive MIP-3αprotein in keratinocytes, melanocytes, dermal microvascular endothelialcells, dermal fibroblast, and dendritic cells in vitro. Furthermore, Thelper cell-derived mediators (e.g., IFN-γ, IL-17, CD40L) regulateMIP-3α production in cellular constituents of the skin. IL-17 is knownto be upregulated in lesional psoriatic skin, suggesting that it mayplay a role in the amplification and the development of cutaneousinflammation. See Nestle, et al. (1994) J. Clin. Invest. 94:202-209; andTeunissen, et al. (1998) J. Invest. Dermatol. 111:645-649. Here it isshown that it is another inducer of MIP-3α protein production by primarykeratinocytes.

[0161] In vitro epithelial Langerhans-type dendritic cells can begenerated from CD34+ hematopoietic progenitor cells, however,monocyte-derived dendritic cells share phenotypic characteristics withdermal dendritic cells. It has been reported previously that CCR6 ishighly expressed on dendritic cells derived from CD34+ hematopoeticprogenitor cells and that MIP-3α selectively induces migratory responsesin CD34+ hematopoetic progenitor cell—but not in monocyte-deriveddendritic cells. See Greaves, et al. (1997) J. Exp. Med. 186:837-844;and Dieu, et al. (1998) J. Exp. Med. 188:373-386. In lesional psoriaticskin, large numbers of dermal dendritic cells are present and showpotent stimulatory functions. Nestle, et al. (1994) J. Clin. Invest.94:202-209. Activation of dendritic cells via CD40 triggering resultedin a marked upregulation of MIP-3α suggesting that dendritic cell-Tlymphocyte interactions may amplify inflammatory-processes in psoriasis.

[0162] In summary, these data suggest that T cell-derived mediators,such as CD40L, IFN-γ, and IL-17, may amplify chemokine, particularlyMIP-3α, production in lesional psoriatic skin and may contribute to thedevelopment and/or chronicity of psoriatic lesions. Thus, blockage ofeffect may be therapeutically effective, e.g., by use of antagonists,receptor antagonists, or receptor desensitization compounds.

[0163] Along with its expression in intestinal epithelial cells,cutaneous MIP-3α expression supports the hypothesis that thisinflammatory chemokine plays an important role in the interface betweenthe organism and the environment. Tanaka, et al. (1999) Eur. J. Immunol.29:633-642. Other chemokines have been shown to be associated withpsoriasis, including RANTES which has been reported to be expressed inpsoriatic lesions by activated keratinocytes. See Fukuoka, et al. (1998)Br. J. Dermatol. 138:63-70; and Raychaudhuri, et al. (1999) Acta Derm.Venereol. 79:9-11. However, peak levels of RANTES expression (2.072ng/ml) in activated keratinocytes were 10-50 times lower than thosedetected for MIP-3α in the present study. Fukuoka, et al. (1998) Br. J.Dermatol. 138:63-70. Furthermore, there is divergent data regardingRANTES expression in lesional psoriatic skin. Fukuoka, et al. (n=3) aswell as Raychaudhuri, et al. (n=8) reported increased RANTES expressionin lesional psoriatic skin using immunohistochemistry in a total of 11psoriatic patients, whereas Goebler, et al. reported no detectableRANTES expression in either lesional psoriatic (n=11), non-lesionalpsoriatic (n=11), or normal skin (n=5). Goebeler, et al. (1998) J.Pathol. 184:89-95. The present study observed markedly lower levels ofRANTES expression in a cDNA library derived from psoriatic skin whencompared with one derived from normal skin using RANTES-specific primersand probes for real time quantitative PCR (TaqMan®) analyses. Incontrast to RANTES, Goebler, et al. detect strong selective expressionof Mig in the upper lesional dermis with pronounced clustering in thetips of the papillae, whereas expression in normal or non-lesionalpsoriatic or normal skin was quiescent. Co-localization studies havesuggested that highly activated dermal macrophages and dermalmicrovascular endothelial cells are major sources of Mig in lesionalpsoriatic skin. Goebeler, et al. (1998) J. Pathol. 184:89-95. Thepro-inflammatory CC chemokines, IP-10 and Mig, are able to attractactivated T cells and are mainly regulated by T cell-derived cytokines,such as IFN-γ. Thus, skin-infiltrating activated T cells releaseinflammatory mediators which, in turn, induce Mig, IP-10, and MIP-3α,contributing to the amplification of inflammatory responses and to thechronicity of psoriatic lesions. Moreover, MCP-1 expression ofkeratinocytes in the stratum basale of lesional psoriatic skin isassociated with chemoattraction of dermal macrophages to lesional sites.See Gillitzer, et al. (1993) J. Invest. Dermatol. 101:127-131; Goebeler,et al. (1998) J. Pathol. 184:89-95; and Deleuran, et al. (1996) J.Dermatol. Sci. 13:228-236.

[0164] Given the cumulating evidence that psoriasis is a T-cell mediateddisease, MIP-3α/CCR6 is the first ligand/receptor pair identified inthis disease which is directly associated with memory T cell recruitmentto lesional psoriatic skin. The only other chemokine/receptor pairsreported in psoriasis, such as IL-8 and GRO-α with their receptors CXCR1and CXCR2, are mainly involved in the recruitment of neutrophils tolesional psoriatic skin. See Gillitzer, et al. (1996) J. Invest.Dermatol. 107:778-782; Gillitzer, et al. (1991) J. Invest. Dermatol.97:73-79; and Kulke, et al. (1996) J. Invest. Dermatol. 106:526-530. Inaddition, the expression pattern of those CXC chemokines did not fullycoincide with the pattern of T cell accumulation. See Gillitzer, et al.(1996) J. Invest. Dermatol. 107:778-782; Gillitzer, et al. (1991) J.Invest. Dermatol. 97:73-79; and Kulke, et al. (1996) J. Invest.Dermatol. 106:526-530.

[0165] Recently, Campbell, et al. suggested that the interaction of theCC chemokine TARC with its receptor CCR4 plays an important role in thehoming of memory T cells to the skin. Campbell, et al. (1999) Nature400:776-780. TARC expression was shown to be exclusively expressed inendothelial cells of both normal and inflamed human skin and this CCchemokine could induce firm adhesion and chemotaxis of skin-homingmemory T cells. Taken together, these findings suggest that TARC andCCR4 may play a role in the interaction of skin-homing T cells with thedermal endothelium and transendothelial migration, however, otherchemokines (e.g., MIP-3α, IP-10, Mig) expressed by keratinocytes,fibroblasts, or dendritic cells may mediate the localization ofskin-homing T cells to the dermis and epidermis. MIP-3α has also beenshown to mediate firm adhesion of CD4+ memory T cells suggesting thatchemokines may also act in an orchestrated fashion at this particularstep of leukocyte trafficking.

[0166] Most recently, a non-chemokine ligand for CCR6 has beenidentified. Yang, et al. showed that human β-defensin-2 is able to bindCCR6 transfected cells and to induce chemotaxis, however, itschemotactic activity was considerably lower than that of MIP-3α. Yang,et al. (1999) [In Process Citation] Science 286:525-528. Interestingly,like MIP-3α, human β-defensin-2 is upregulated by proinflammatorymediators, such as TNF-α and IL-1β. Most importantly, bacterial andviral infections markedly induce the production of human β-defensin-2.Since both MIP-3α and human β-defensin-2 are expressed in psoriaticskin, they could both contribute to the recruitment of memory T cells tolesional sites, however, these studies found no evidence for a role forhuman β-defensin-2 since the intracellular Ca² ^(₊) mobilization inducedby supernatants of TNF-α/IL-1β-stimulated primary keratinocytes in CCR6transfectants was completely blocked by an anti-MIP-3α antibody.

[0167] These findings suggest the following model for the involvement ofMIP-3α and CCR6 in the pathogenesis of psoriasis: MIP-3α may be inducedin keratinocytes and/or dermal microvascular endothelial cells at sitesof physical injury or infection due to the release of proinflammatorycytokines, such as TNF-α and IL-1. In turn, TARC and MIP-3α may induceadhesion (Campbell, et al. (1998) Science 279:381-384; and Campbell, etal. (1999) Nature 400:776-780) and chemotaxis of skin-homing memory Tcells (Liao, et al. (1999) J. Immunol. 162:186-194) through theendothelium into the skin. Subsequently, the skin-homing CLA+ T cellsmay encounter their specific antigen presented by dendritic cells, getactivated, and produce inflammatory mediators, such as IFN-γ, IL-17, orCD40L, which, in turn, induce additional MIP-3α, IP-10, and Migproduction by activated keratinocytes, dendritic cells, and dermalmacrophages. Gottlieb, et al. (1988) J. Exp. Med. 168:941-948; Goebeler,et al. (1998) J. Pathol. 184:89-95; and Boorsma, et al. (1998) Arch.Dermatol. Res. 290:335-341. This “second wave” of chemokine productionmay complete a self-sustaining cycle of inflammation which leads to thedevelopment of a psoriatic phenotype.

[0168] In conclusion, this study shows the potential that a highlyspecific and sensitive real time quantitative PCR technique (TaqMan® )offers to identify novel disease associations with the expression ofspecific genes. This technology led to the identification of MIP-3α/CCR6as a new ligand/receptor pair potentially involved in the pathogenesisof psoriasis.

[0169] All citations herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

[0170] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

1 8 1 291 DNA Homo sapiens CDS (1)..(288) 1 atg tgc tgt acc aag agt ttgctc ctg gct gct ttg atg tca gtg ctg 48 Met Cys Cys Thr Lys Ser Leu LeuLeu Ala Ala Leu Met Ser Val Leu -25 -20 -15 cta ctc cac ctc tgc ggc gaatca gaa gca gca agc aac ttt gac tgc 96 Leu Leu His Leu Cys Gly Glu SerGlu Ala Ala Ser Asn Phe Asp Cys -10 -5 -1 1 5 tgt ctt gga tac aca gaccgt att ctt cat cct aaa ttt att gtg ggc 144 Cys Leu Gly Tyr Thr Asp ArgIle Leu His Pro Lys Phe Ile Val Gly 10 15 20 ttc aca cgg cag ctg gcc aatgaa ggc tgt gac atc aat gct atc atc 192 Phe Thr Arg Gln Leu Ala Asn GluGly Cys Asp Ile Asn Ala Ile Ile 25 30 35 ttt cac aca aag aaa aag ttg tctgtg tgc gca aat cca aaa cag act 240 Phe His Thr Lys Lys Lys Leu Ser ValCys Ala Asn Pro Lys Gln Thr 40 45 50 tgg gtg aaa tat att gtg cgt ctc ctcagt aaa aaa gtc aag aac atg 288 Trp Val Lys Tyr Ile Val Arg Leu Leu SerLys Lys Val Lys Asn Met 55 60 65 70 taa 291 2 96 PRT Homo sapiens 2 MetCys Cys Thr Lys Ser Leu Leu Leu Ala Ala Leu Met Ser Val Leu -25 -20 -15Leu Leu His Leu Cys Gly Glu Ser Glu Ala Ala Ser Asn Phe Asp Cys -10 -5-1 1 5 Cys Leu Gly Tyr Thr Asp Arg Ile Leu His Pro Lys Phe Ile Val Gly10 15 20 Phe Thr Arg Gln Leu Ala Asn Glu Gly Cys Asp Ile Asn Ala Ile Ile25 30 35 Phe His Thr Lys Lys Lys Leu Ser Val Cys Ala Asn Pro Lys Gln Thr40 45 50 Trp Val Lys Tyr Ile Val Arg Leu Leu Ser Lys Lys Val Lys Asn Met55 60 65 70 3 291 DNA Mus musculus CDS (1)..(288) 3 atg gcc tgc ggt ggcaag cgt ctg ctc ttc ctt gct ttg gca tgg gta 48 Met Ala Cys Gly Gly LysArg Leu Leu Phe Leu Ala Leu Ala Trp Val -25 -20 -15 ctg ctg gct cac ctctgc agc cag gca gaa gca agc aac tac gac tgt 96 Leu Leu Ala His Leu CysSer Gln Ala Glu Ala Ser Asn Tyr Asp Cys -10 -5 -1 1 5 tgc ctc tcg tacata cag acg cca ctt cct tcc aga gct att gtg ggt 144 Cys Leu Ser Tyr IleGln Thr Pro Leu Pro Ser Arg Ala Ile Val Gly 10 15 20 ttc aca aga cag atggcc gat gaa gct tgt gac att aat gct atc atc 192 Phe Thr Arg Gln Met AlaAsp Glu Ala Cys Asp Ile Asn Ala Ile Ile 25 30 35 ttt cac acg aag aaa agaaaa tct gtg tgc gct gat cca aag cag aac 240 Phe His Thr Lys Lys Arg LysSer Val Cys Ala Asp Pro Lys Gln Asn 40 45 50 tgg gtg aaa agg gct gtg aacctc ctc agc cta aga gtc aag aag atg 288 Trp Val Lys Arg Ala Val Asn LeuLeu Ser Leu Arg Val Lys Lys Met 55 60 65 taa 291 4 96 PRT Mus musculus 4Met Ala Cys Gly Gly Lys Arg Leu Leu Phe Leu Ala Leu Ala Trp Val -25 -20-15 Leu Leu Ala His Leu Cys Ser Gln Ala Glu Ala Ser Asn Tyr Asp Cys -10-5 -1 1 5 Cys Leu Ser Tyr Ile Gln Thr Pro Leu Pro Ser Arg Ala Ile ValGly 10 15 20 Phe Thr Arg Gln Met Ala Asp Glu Ala Cys Asp Ile Asn Ala IleIle 25 30 35 Phe His Thr Lys Lys Arg Lys Ser Val Cys Ala Asp Pro Lys GlnAsn 40 45 50 Trp Val Lys Arg Ala Val Asn Leu Leu Ser Leu Arg Val Lys LysMet 55 60 65 5 291 DNA Rattus sp. CDS (1)..(288) 5 atg gcc tgc aag catctg ccc ttc ctg gct ttg gcg ggg gta ctg ctg 48 Met Ala Cys Lys His LeuPro Phe Leu Ala Leu Ala Gly Val Leu Leu -25 -20 -15 -10 gct tac ctc tgcagc cag tca gaa gca gca agc aac ttt gac tgc tgc 96 Ala Tyr Leu Cys SerGln Ser Glu Ala Ala Ser Asn Phe Asp Cys Cys -5 -1 1 5 ctc acg tac acaaag aac gtg tat cat cat gcg aga aat ttt gtg ggt 144 Leu Thr Tyr Thr LysAsn Val Tyr His His Ala Arg Asn Phe Val Gly 10 15 20 ttc aca aca cag atggcc gac gaa gct tgt gac att aat gct atc atc 192 Phe Thr Thr Gln Met AlaAsp Glu Ala Cys Asp Ile Asn Ala Ile Ile 25 30 35 ttt cac ctg aag tcg aaaaga tcc gtg tgc gct gac cca aag cag atc 240 Phe His Leu Lys Ser Lys ArgSer Val Cys Ala Asp Pro Lys Gln Ile 40 45 50 55 tgg gtg aaa agg att ttgcac ctc ctc agc cta aga acc aag aag atg 288 Trp Val Lys Arg Ile Leu HisLeu Leu Ser Leu Arg Thr Lys Lys Met 60 65 70 taa 291 6 96 PRT Rattus sp.6 Met Ala Cys Lys His Leu Pro Phe Leu Ala Leu Ala Gly Val Leu Leu -25-20 -15 -10 Ala Tyr Leu Cys Ser Gln Ser Glu Ala Ala Ser Asn Phe Asp CysCys -5 -1 1 5 Leu Thr Tyr Thr Lys Asn Val Tyr His His Ala Arg Asn PheVal Gly 10 15 20 Phe Thr Thr Gln Met Ala Asp Glu Ala Cys Asp Ile Asn AlaIle Ile 25 30 35 Phe His Leu Lys Ser Lys Arg Ser Val Cys Ala Asp Pro LysGln Ile 40 45 50 55 Trp Val Lys Arg Ile Leu His Leu Leu Ser Leu Arg ThrLys Lys Met 60 65 70 7 1098 DNA Homo sapiens CDS (1)..(1095) 7 atg ttttcg act cca gtg aag att att ttg tgt cag tca ata ctt cat 48 Met Phe SerThr Pro Val Lys Ile Ile Leu Cys Gln Ser Ile Leu His 1 5 10 15 att actcag ttg att ctg aga tgt tac tgt gct cct tgc agg agg tca 96 Ile Thr GlnLeu Ile Leu Arg Cys Tyr Cys Ala Pro Cys Arg Arg Ser 20 25 30 ggc agt tctcca ggc tat ttg tac cga att gcc tac tcc ttg atc tgt 144 Gly Ser Ser ProGly Tyr Leu Tyr Arg Ile Ala Tyr Ser Leu Ile Cys 35 40 45 gtt ctt ggc ctcctg ggg aat att ctg gtg gtg atc acc ttt gct ttt 192 Val Leu Gly Leu LeuGly Asn Ile Leu Val Val Ile Thr Phe Ala Phe 50 55 60 tat aag aag gcc aggtct atg aca gac gtc tat ctc ttg aac atg gcc 240 Tyr Lys Lys Ala Arg SerMet Thr Asp Val Tyr Leu Leu Asn Met Ala 65 70 75 80 att gca gac atc ctcttt gtt ctt act ctc cca ttc tgg gca gtg agt 288 Ile Ala Asp Ile Leu PheVal Leu Thr Leu Pro Phe Trp Ala Val Ser 85 90 95 cat gcc act ggt gcg tgggtt ttc agc aat gcc acg tgc aag ttg cta 336 His Ala Thr Gly Ala Trp ValPhe Ser Asn Ala Thr Cys Lys Leu Leu 100 105 110 aaa ggc atc tat gcc atcaac ttt aac tgc ggg atg ctg ctc ctg act 384 Lys Gly Ile Tyr Ala Ile AsnPhe Asn Cys Gly Met Leu Leu Leu Thr 115 120 125 tgc att agc atg gac cggtac atc gcc att gta cag gcg act aag tca 432 Cys Ile Ser Met Asp Arg TyrIle Ala Ile Val Gln Ala Thr Lys Ser 130 135 140 ttc cgg ctc cga tcc agaaca cta ccg cgc agc aaa atc atc tgc ctt 480 Phe Arg Leu Arg Ser Arg ThrLeu Pro Arg Ser Lys Ile Ile Cys Leu 145 150 155 160 gtt gtg tgg ggg ctgtca gtc atc atc tcc agc tca act ttt gtc ttc 528 Val Val Trp Gly Leu SerVal Ile Ile Ser Ser Ser Thr Phe Val Phe 165 170 175 aac caa aaa tac aacacc caa ggc agc gat gtc tgt gaa ccc aag tac 576 Asn Gln Lys Tyr Asn ThrGln Gly Ser Asp Val Cys Glu Pro Lys Tyr 180 185 190 can act gtc tcg gagccc atc agg tgg aag ctg ctg atg ttg ggg ctt 624 Thr Thr Val Ser Glu ProIle Arg Trp Lys Leu Leu Met Leu Gly Leu 195 200 205 gag cta ctc ttt ggtttc ttt atc cct ttg atg ttc atg ata ttt tgt 672 Glu Leu Leu Phe Gly PhePhe Ile Pro Leu Met Phe Met Ile Phe Cys 210 215 220 tac acg ttc att gtcaaa acc ttg gtg caa gct cag aat tct aaa agg 720 Tyr Thr Phe Ile Val LysThr Leu Val Gln Ala Gln Asn Ser Lys Arg 225 230 235 240 cac aaa gcc atccgt gta atc ata gct gtg gtg ctt gtg ttt ctg gct 768 His Lys Ala Ile ArgVal Ile Ile Ala Val Val Leu Val Phe Leu Ala 245 250 255 tgt cag att cctcat aac atg gtc ctg ctt gtg acg gct gct aat ttg 816 Cys Gln Ile Pro HisAsn Met Val Leu Leu Val Thr Ala Ala Asn Leu 260 265 270 ggt aaa atg aaccga tcc tgc cag agc gaa aag cta att ggc tat acg 864 Gly Lys Met Asn ArgSer Cys Gln Ser Glu Lys Leu Ile Gly Tyr Thr 275 280 285 aaa act gtc acagaa gtc ctg gct ttc ctg cac tgc tgc ctg aac cct 912 Lys Thr Val Thr GluVal Leu Ala Phe Leu His Cys Cys Leu Asn Pro 290 295 300 gtg ctc tac gctttt att ggg cag aag ttc aga aac tac ttt ctg aag 960 Val Leu Tyr Ala PheIle Gly Gln Lys Phe Arg Asn Tyr Phe Leu Lys 305 310 315 320 atc ttg aaggac ctg tgg tgt gtg aga agg aag tac aag tcc tca ggc 1008 Ile Leu Lys AspLeu Trp Cys Val Arg Arg Lys Tyr Lys Ser Ser Gly 325 330 335 ttc tcc tgtgcc ggg agg tac tca gaa aac att tct cgg cag acc agt 1056 Phe Ser Cys AlaGly Arg Tyr Ser Glu Asn Ile Ser Arg Gln Thr Ser 340 345 350 gag acc gcagat aac gac aat gcg tcg tcc ttc act atg tga 1098 Glu Thr Ala Asp Asn AspAsn Ala Ser Ser Phe Thr Met 355 360 365 8 365 PRT Homo sapiensmisc_feature (579)..(579) n may be a, c, g, or t 8 Met Phe Ser Thr ProVal Lys Ile Ile Leu Cys Gln Ser Ile Leu His 1 5 10 15 Ile Thr Gln LeuIle Leu Arg Cys Tyr Cys Ala Pro Cys Arg Arg Ser 20 25 30 Gly Ser Ser ProGly Tyr Leu Tyr Arg Ile Ala Tyr Ser Leu Ile Cys 35 40 45 Val Leu Gly LeuLeu Gly Asn Ile Leu Val Val Ile Thr Phe Ala Phe 50 55 60 Tyr Lys Lys AlaArg Ser Met Thr Asp Val Tyr Leu Leu Asn Met Ala 65 70 75 80 Ile Ala AspIle Leu Phe Val Leu Thr Leu Pro Phe Trp Ala Val Ser 85 90 95 His Ala ThrGly Ala Trp Val Phe Ser Asn Ala Thr Cys Lys Leu Leu 100 105 110 Lys GlyIle Tyr Ala Ile Asn Phe Asn Cys Gly Met Leu Leu Leu Thr 115 120 125 CysIle Ser Met Asp Arg Tyr Ile Ala Ile Val Gln Ala Thr Lys Ser 130 135 140Phe Arg Leu Arg Ser Arg Thr Leu Pro Arg Ser Lys Ile Ile Cys Leu 145 150155 160 Val Val Trp Gly Leu Ser Val Ile Ile Ser Ser Ser Thr Phe Val Phe165 170 175 Asn Gln Lys Tyr Asn Thr Gln Gly Ser Asp Val Cys Glu Pro LysTyr 180 185 190 Thr Thr Val Ser Glu Pro Ile Arg Trp Lys Leu Leu Met LeuGly Leu 195 200 205 Glu Leu Leu Phe Gly Phe Phe Ile Pro Leu Met Phe MetIle Phe Cys 210 215 220 Tyr Thr Phe Ile Val Lys Thr Leu Val Gln Ala GlnAsn Ser Lys Arg 225 230 235 240 His Lys Ala Ile Arg Val Ile Ile Ala ValVal Leu Val Phe Leu Ala 245 250 255 Cys Gln Ile Pro His Asn Met Val LeuLeu Val Thr Ala Ala Asn Leu 260 265 270 Gly Lys Met Asn Arg Ser Cys GlnSer Glu Lys Leu Ile Gly Tyr Thr 275 280 285 Lys Thr Val Thr Glu Val LeuAla Phe Leu His Cys Cys Leu Asn Pro 290 295 300 Val Leu Tyr Ala Phe IleGly Gln Lys Phe Arg Asn Tyr Phe Leu Lys 305 310 315 320 Ile Leu Lys AspLeu Trp Cys Val Arg Arg Lys Tyr Lys Ser Ser Gly 325 330 335 Phe Ser CysAla Gly Arg Tyr Ser Glu Asn Ile Ser Arg Gln Thr Ser 340 345 350 Glu ThrAla Asp Asn Asp Asn Ala Ser Ser Phe Thr Met 355 360 365

What is claimed is:
 1. A method of modulating migration of a cell withinor to the skin of a mammal, said method comprising administering to saidmammal an effective amount of: a) an antagonist of MIP-3α; b) an agonistof MIP-3α; c) an antagonist of CCR6; or d) an agonist of CCR6.
 2. Themethod of claim 1, wherein said migration is within said skin.
 3. Themethod of claim 2, wherein said migration is chemotactic orchemokinetic.
 4. The method of claim 1, wherein said administering issystemic, local, topical, subcutaneous, intracutaneous, or transdermal.5. The method of claim 1, wherein said cell is a T cell, B cell,dendritic cell, or dendritic cell precursor.
 6. The method of claim 5,wherein said cell is a T cell.
 7. The method of claim 1, wherein saidcell migrates into the dermal and/or epidermal layers of said skin. 8.The method of claim 1, wherein said administering is an antagonist ofMIP-3α.
 9. The method of claim 8, wherein said antagonist is selectedfrom: a) a mutein of natural MIP-3α; b) an antibody which neutralizesMIP-3α; or c) an antibody which binds to CCR6.
 10. The method of claim8, wherein said mammal is subject to a skin condition, including oneselected from cancer, cancer metastasis, autoimmunity, inflamation,infection, psoriasis, skin transplant, or skin graft.
 11. The method ofclaim 8, wherein said antagonist is administered in combination with anantibiotic, antifungal, antiviral, cancer therapy, or analgesic.
 12. Themethod of claim 8, wherein said antagonist is administered incombination with an immune suppressive therapeutic, anti-inflammatorydrug, growth factor, or immune adjuvant.
 13. The method of claim 1,wherein said administering is with a primate MIP-3α.
 14. The method ofclaim 13, wherein said modulating is attracting said cell.
 15. Themethod of claim 14, wherein said cell is attracted to a site ofcutaneous lesion.
 16. The method of claim 13, wherein said primateMIP-3α is administered in combination with an antibiotic, antifungal,antiviral, or analgesic.
 17. The method of claim 13, wherein said MIP-3αis administered in combination with a vasodilator, growth factor,cytokine, anti-inflammatory drug, or immune adjuvant.
 18. A method ofpurifying a population of cells, said method comprising contacting saidcells with MIP-3α, thereby resulting in the identification of cellsexpressing a receptor for said MIP-3α.
 19. The method of claim 18,wherein: a) said receptor is CCR6; or b) said contacting results inspecific migration of said cells to a site for purification.
 20. Themethod of claim 18, wherein said migration is through pores of amembrane.